Postpartum cells derived from umbilical cord tissue, and methods of making and using the same

ABSTRACT

Cells derived from human umbilical cords are disclosed along with methods for their therapeutic use. Isolation techniques, culture methods and detailed characterization of the cells with respect to their cell surface markers, gene expression, and their secretion of trophic factors are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/877,012, filed Jun. 25, 2004, which in turn claims benefit of U.S.Provisional Application Ser. No. 60/483,264, filed Jun. 27, 2003. Theentire contents of these prior applications are incorporated byreference herein for all purposes. Other related applications includethe following commonly-owned, co-pending applications, the entirecontents of each of which are incorporated by reference herein: U.S.Application No. [ETH-5073 NP2], filed Jun. 25, 2004; U.S. ApplicationNo. [ETH-5073 NP3], filed Jun. 25, 2004; U.S. Application No. [ETH-5073NP4], filed Jun. 25, 2004; U.S. Application No. [ETH-5073 NP5], filedJun. 25, 2004; U.S. Application No. [ETH-5073 NP7], filed Jun. 25, 2004;U.S. Application No. [ETH-5073 NP8], filed Jun. 25, 2004; and U.S.Provisional Application No. 60/555,908, filed Mar. 24, 2004.

FIELD OF THE INVENTION

This invention relates to mammalian, preferably human, cell therapy, andmore particularly to isolated cells derived from postpartum umbilicus,methods of deriving such cells, and methods of their use.

BACKGROUND OF THE INVENTION

As the modern understanding of disease has advanced, the potentialutility of cell therapy for improving the prognosis of those afflictedhas resulted in increased interest in new sources of human cells usefulfor therapeutic purposes. One such source of human cells is postpartumtissues, and in particular, the umbilicus or umbilical cord.

Recently, attention has focused on the banking of umbilical cord blood(or simply “cord blood”) as a potential source of, for example,hematapoeitic cells for use by an individual for whom cord blood hasbeen banked at birth. Such cells would be useful for those individuals,for example, who require therapeutic radiation which may eliminatefunctional portions of their immune system. Rather than requiring a bonemarrow donor carefully matched to avoid rejection, the individual's ownbanked cord blood could be used to reconstitute the lost immune cells,and restore immune function.

Still more recently, there has been interest in obtaining stem cellsfrom cord blood, due to the wider potential therapeutic applications ofsuch cells. Stem cells are understood in general terms as cells that 1)have the ability to self-renew for long periods through cell divisionfrom a single cell; and 2) have the ability to differentiate intospecific cell types given the proper conditions. Accordingly, stem cellsare potentially useful in treating a population of individuals, and notmerely the person from whose cord blood the cells were initiallyobtained.

In particular, cord blood has been considered as a source ofhematopoietic progenitor stem cells. Banked (or cryopreserved) cordblood, or stem cells isolated therefrom have been deemed useful forhematopoietic reconstitution, for example in bone marrow and relatedtransplantations. (Boyse et al., U.S. Pat. Nos. 5,004,681 and5,192,553).

In addition to cord blood, other sources of therapeutic cells from thehuman umbilicus have been explored, including cells isolated from theWharton's Jelly, umbilical vein or artery tissue, and the umbilicalmatrix itself. Such cells have been largely uncharacterized, or onlyminimally characterized with respect to their physiological,biochemical, immunological, and genetic properties.

For example, Purchio et al. (U.S. Pat. No. 5,919,702) have isolatedchondrogenic progenitor cells (or prechondrocytes) from Wharton's Jelly.They reported the isolation of cells from human umbilical cord Wharton'sJelly by removing blood and blood vessels and incubating the tissueunder conditions purported to allow the prechodrocytes to proliferate.As such, the method did not distinguish the desired cells from thedifferent cell types present in Wharton's Jelly, but rather relied onmigration from the tissue or selecting growth conditions favoringprechondrocytes. The prechondrocytes were expanded mitotically afterthey were established. Cells at passages 2 to 4 were reported as usefulto produce cartilage, if triggered by the addition of exogenous growthfactors, such as BMP-13 or TGF-beta. Uses of the cells for directinjection or implantation, or use with a hydrogel or tissue matrix, wereproposed. However, it was considered important that the cells not exceedabout 25% confluence. The cells were not characterized with respect totheir biochemical or immunological properties, or with respect to theirgene expression.

Weiss et al. (U.S. Patent Application Publication US2003/0161818)proposed procedures for isolating pluripotent or lineage-committed cellsfrom mammalian Wharton's Jelly or non-blood umbilical cord matrixsources. The cells isolated were reported to differentiate intohematopoietic, mesenchymal or neuroectodermal lines. The cell lines werenot characterized with respect to their identifying properties. Limitedcharacterization was provided with respect to cells afterdifferentiation towards neural lines. Reference was made to Wharton'sJelly-derived bovine and porcine cells that were CD34⁻, CD45⁻.

Weiss et al. also reported investigating transplantation of porcineumbilical cord matrix cells into rat brain. (Exp. Neur. 182: 288-299,2003). No enzymatic treatment was used in the isolation procedure. Theyobtained two distinct populations—spherical and flat mesenchymal cells.The cells were genetically modified to express GFP. The cells did notappear to stimulate immune rejection when implanted cross-species.

Mitchell et al. (Stem Cells 21:50-60, 2003) reported obtaining Wharton'sJelly matrix cells from porcine umbilical cords. The undifferentiatedcells were reported to be positive for telomerase and a subpopulationwas also reported positive for c-kit expression, i.e., telomerase⁺,CD117⁺. The cells were also reported to produce alpha-smooth muscleactin, indicative of their myofibroblast-like nature. The cellspurportedly could differentiate along neural lines in the presence ofgrowth factors. However, both the differentiated and undifferentiatedcells were found to express NSE, a marker for neural stem cells. Theneed for clonal lines and characterization in terms of proliferativecapacity, karyotype analysis, and expression of HLA antigens wasrecognized.

Romanov et al. (Stem Cells 21(1):105-110, 2003) reported a procedure toisolate mesenchymal stem cell (MSC)-like cells from human umbilical cordvein. Their procedure involved treatment of the excised vein tissue withcollagenase and required that the enzymatic digestion be short (15minutes) to obtain the cell population of interest (a subendotheliallayer of the vein). In particular, the procedure avoided inclusion ofsmooth muscle cells (SMCs) and fibroblasts by leaving the deeper layersof the tissue intact, purportedly removing only the outer layers. Theresulting “nearly homogenous” cell population was reported to containapproximately 0.5-1% endothelial cells that they also sought to avoid.The cells were reported to be predominantly CD34⁻ and to producealpha-smooth muscle actin.

Because of the diversity of cell populations that are found in umbilicalcord matrix, there is a need in the art for methods of isolating definednon-blood cells and populations thereof derived from mammalian umbilicalcord; as well as a need for cell lines derived from mammalian umbilicusthat are characterized with respect to their biochemistry (e.g.secretion of growth factors), immunology (cell surface markers andpotential to stimulate immune responses) and expression of variousgenes. This need is particularly compelling for cells derived from humanumbilicus.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides isolated umbilicus-derivedcells (UDCs) that are derived from mammalian umbilical cord tissuesubstantially free of blood. The cells are capable of self-renewal andexpansion in culture. The umbilicus-derived cells have the potential todifferentiate into cells of other phenotypes. In preferred embodiments,the cells are derived from human umbilicus.

The cells have been characterized as to several of their cellular,genetic, immunological, and biochemical properties. For example, thecells have been characterized by their growth properties in culture, bytheir cell surface markers, by their gene expression, by their abilityto produce certain biochemical trophic factors, and by theirimmunological properties.

In certain embodiments, the postpartum-derived cell is anumbilicus-derived cell. In other embodiments it is a placenta-derivedcell. In specific embodiments, the cell has all identifying features ofeither of cell types UMB 022803 (P7) (ATCC Accession No. PTA-6067); orUMB 022803 (P17) (ATCC Accession No. PTA-6068).

In another of its several aspects, cell cultures comprising the isolatedumbilicus-derived cells of the invention are provided. The cultures ofumbilical cells are free of maternal cells in certain preferredembodiments.

Methods of culturing and expanding umbilicus-derived cells and cellcultures and populations comprising them are provided.

In another aspect of the invention isolated umbilicus-derived cellshaving specific cell surface marker expression profiles are provided,wherein particular cell surface marker proteins are produced. Inparticular, the cells produce one or more of CD10, CD13, CD44, CD73,CD90, CD141, PDGFr-alpha, or HLA-A,B,C. In addition, the cells do notproduce one or more of CD31, CD34, CD45, CD117, CD141, or HLA-DR,DP,DQ,as detected by flow cytometry.

The cells of the invention have also been characterized according totheir expression of a wide variety of genes. Accordingly, another aspectof the invention provides isolated umbilicus-derived cells, whichrelative to a human cell that is a fibroblast, a mesenchymal stem cell,or an ileac crest bone marrow cell, have reduced expression of genes forone or more of: short stature homeobox 2; heat shock 27 kDa protein 2;chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);elastin (supravalvular aortic stenosis, Williams-Beuren syndrome); Homosapiens mRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeobox 2 (growth arrest-specific homeobox); sine oculis homeoboxhomolog 1 (Drosophila); crystallin, alpha B; disheveled associatedactivator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin1; tetranectin (plasminogen binding protein); src homology three (SH3)and cysteine rich domain; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; interleukin 11 receptor, alpha; procollagenC-endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypotheticalgene BC008967; collagen, type VIII, alpha 1; tenascin C (hexabrachion);iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; neuroblastoma, suppression of tumorigenicity 1;insulin-like growth factor binding protein 2, 36 kDa; Homo sapiens cDNAFLJ12280 fis, clone MAMMA1001744; cytokine receptor-like factor 1;potassium intermediate/small conductance calcium-activated channel,subfamily N, member 4; integrin, beta 7; transcriptional co-activatorwith PDZ-binding motif (TAZ); sine oculis homeobox homolog 2(Drosophila); KIAA1034 protein; vesicle-associated membrane protein 5(myobrevin); EGF-containing fibulin-like extracellular matrix protein 1;early growth response 3; distal-less homeobox 5; hypothetical proteinFLJ20373; aldo-keto reductase family 1, member C3 (3-alphahydroxysteroid dehydrogenase, type II); biglycan; transcriptionalco-activator with PDZ-binding motif (TAZ); fibronectin 1; proenkephalin;integrin, beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNAfull length insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367protein; natriuretic peptide receptor C/guanylate cyclase C(atrionatriuretic peptide receptor C); hypothetical protein FLJ14054;Homo sapiens mRNA; cDNA DKFZp564B222 (from clone DKFZp564B222);BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; and cytochrome c oxidase subunit VIIa polypeptide 1 (muscle).In addition, these isolated human umbilicus-derived cells express a genefor each of interleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1(melonoma growth stimulating activity, alpha); chemokine (C-X-C motif)ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-C motif)ligand 3; and tumor necrosis factor, alpha-induced protein 3, whereinthe expression is increased relative to that of a human cell which is afibroblast, a mesenchymal stem cell, an ileac crest bone marrow cell, orplacenta-derived cell. The cells are capable of self-renewal andexpansion in culture, and have the potential to differentiate into cellsof other phenotypes. Also provided are therapeutic cell culturescomprising the isolated human umbilicus-derived cells.

In another of its several aspects, the invention provides isolated humanumbilicus-derived cells capable of self-renewal and expansion in cultureand which have the potential to differentiate into cells of otherphenotypes, wherein the cells do not stimulate allogeneic lymphocytes ina mixed lymphocyte reaction, and expresses PD-L2, but not HLA-G, CD178,HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, or B7-H2. In some preferredembodiments, the cells do not stimulate allogeneic PBMCs. Morepreferably they do not stimulate allogeneic lymphocytes, allogeneicT-cells, or naïve T-cells, or generate other adverse immunologicalresponses in either matched or unmatched recipients. The cells also canproduce vimentin and alpha-smooth muscle actin in certain embodiments.

In another aspect, the invention provides isolated humanumbilicus-derived cells that secrete one or more of the angiogenicfactors MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, orTIMP1. In certain embodiments, the cells secrete several or all of theaforementioned molecules. In other embodiments, the cells do not secreteone or more of the angiogenic factors SDF-1alpha, TGF-beta2, ANG2,PDGFbb or VEGF, as detected by ELISA. In particular embodiments, theysecrete few or none of those molecules.

In another aspect of the invention, therapeutic cell cultures areprovided, the cell cultures comprising the isolated cells as describedabove for use in treating patients in need of angiogenesis-stimulatingtrophic factors. Such therapeutic cell cultures are also provided foruse in treating a patient in need of neural growth stimulating trophicfactors.

Methods of deriving cells from non-blood human umbilical tissue areprovided. The cells are capable of self-renewal and expansion inculture, and have the potential to differentiate into cells of otherphenotypes. The method comprises (a) obtaining human umbilical tissue;(b) removing substantially all of the blood to yield a substantiallyblood-free umbilical tissue, (c) dissociating the tissue by mechanicalor enzymatic treatment, or both, (d) resuspending the tissue in aculture medium, and (e) providing growth conditions which allow for thegrowth of a human umbilicus-derived cell capable of self-renewal andexpansion in culture and having the potential to differentiate intocells of other phenotypes. Preferred methods involve enzymatic treatmentwith, for example, collagenase and dispase, or collagenase, dispase, andhyaluronidase, and such methods are provided herein.

Isolated human umbilicus-derived cells derived by the above method arealso provided herein. The cells maintain a consistent normal karyotypenotwithstanding repeated passaging in certain embodiments. Also providedare cultures of human umbilicus-derived cells derived by the abovemethod, wherein the cultures are free of maternal cells.

Co-cultures comprising the cells or cultures of the invention with othermammalian cells are also provided herein. Preferably these co-culturescomprise another human cell line whose growth or therapeutic potential,for example, is improved by the presence of the umbilicus-derived cells.Such co-cultures are useful for therapeutic application in vitro or invivo.

Also provided herein are therapeutic compositions comprising anumbilicus-derived cell and another therapeutic agent, factor, orbioactive agent. Such factors include, but are not limited to, IGF, LIF,PDGF, EGF, FGF, as well as antithrombogenic, antiapoptotic agents,anti-inflammatory agents, immunosuppressive or immunomodulatory agents,and antioxidants. Such therapeutic compositions can further comprise oneor more additional cell types in addition to the UDCs and the bioactivecomponent.

In addition to the above, compositions derived from the cells areprovided herein. Cell lysates, soluble cell fractions andmembrane-enriched cell fractions are provided herein. Extracellularmatrices derived from the cells, for example, comprising basementmembranes are also useful and are provided herein.

Compositons of the invention also include conditioned culture media asprovided herein. Such media have first been used to grow the cells orcultures of the invention, which during growth secrete one or moreuseful products into the medium. Conditioned medium from these novelcells are useful for many purposes, including for example, supportingthe growth of other mammalian cells in need of growth factors or trophicfactors secreted into the media by the cells and cultures of theinvention, and promoting, for example, angiogenesis.

Methods are provided of inducing the cells to differentiate along apathway towards progenitors of various cells, or even into terminallydifferentiated cells themselves. Such cells have utility for therapeutictreatment of certain conditions, disorders and disease states. Suchcells also have utility for diagnostic protocols, such as for use inassays to identify therapeutic agents.

The invention also provides methods of utilizing the differentiatedumbilicus-derived cells or the progenitors for therapeutic uses,including but not limited to angiogenic application, neuronalapplications, soft tissue applications, occular applications, andapplications wherein the cells are useful in treatment of heart, kidney,bone, cartilage, pancreas, liver, and other tissues alone or incombination with other therapeutic agents.

Kits are also provided herein. Kits useful for the growth, isolation anduse of the umbilical-derived cells are provided.

These and further aspects of the invention will be described in greaterdetail below.

DETAILED DESCRIPTION

Definitions

Various terms used throughout the specification and claims are definedas set forth below.

Stem cells are undifferentiated cells defined by the ability of a singlecell both to self-renew, and to differentiate to produce progeny cells,including self-renewing progenitors, non-renewing progenitors, andterminally differentiated cells. Stem cells are also characterized bytheir ability to differentiate in vitro into functional cells of variouscell lineages from multiple germ layers (endoderm, mesoderm andectoderm), as well as to give rise to tissues of multiple germ layersfollowing transplantation, and to contribute substantially to most, ifnot all, tissues following injection into blastocysts.

Stem cells are classified according to their developmental potential as:(1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and(5) unipotent. Totipotent cells are able to give rise to all embryonicand extraembryonic cell types. Pluripotent cells are able to give riseto all embryonic cell types. Multipotent cells include those able togive rise to a subset of cell lineages, but all within a particulartissue, organ, or physiological system (for example, hematopoietic stemcells (HSC) can produce progeny that include HSC (self-renewal), bloodcell-restricted oligopotent progenitors, and all cell types and elements(e.g., platelets) that are normal components of the blood). Cells thatare oligopotent can give rise to a more restricted subset of celllineages than multipotent stem cells; and cells that are unipotent areable to give rise to a single cell lineage (e.g., spermatogenic stemcells).

Stem cells are also categorized on the basis of the source from whichthey may be obtained. An adult stem cell is generally a multipotentundifferentiated cell found in tissue comprising multiple differentiatedcell types. The adult stem cell can renew itself. Under normalcircumstances, it can also differentiate to yield the specialized celltypes of the tissue from which it originated, and possibly other tissuetypes. An embryonic stem cell is a pluripotent cell from the inner cellmass of a blastocyst-stage embryo. A fetal stem cell is one thatoriginates from fetal tissues or membranes. A postpartum stem cell is amultipotent or pluripotent cell that originates substantially fromextraembryonic tissue available after birth, namely, the placenta andthe umbilical cord. These cells have been found to possess featurescharacteristic of pluripotent stem cells, including rapid proliferationand the potential for differentiation into many cell lineages.Postpartum stem cells may be blood-derived (e.g., as are those obtainedfrom umbilical cord blood) or non-blood-derived (e.g., as obtained fromthe non-blood tissues of the umbilical cord and placenta).

Embryonic tissue is typically defined as tissue originating from theembryo (which in humans refers to the period from fertilization to aboutsix weeks of development. Fetal tissue refers to tissue originating fromthe fetus, which in humans refers to the period from about six weeks ofdevelopment to parturition. Extraembryonic tissue is tissue associatedwith, but not originating from, the embryo or fetus. Extraembryonictissues include extraembryonic membranes (chorion, amnion, yolk sac andallantois), umbilical cord and placenta (which itself forms from thechorion and the maternal decidua basalis).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cell,such as a nerve cell or a muscle cell, for example. A differentiatedcell is one that has taken on a more specialized (“committed”) positionwithin the lineage of a cell. The term committed, when applied to theprocess of differentiation, refers to a cell that has proceeded in thedifferentiation pathway to a point where, under normal circumstances, itwill continue to differentiate into a specific cell type or subset ofcell types, and cannot, under normal circumstances, differentiate into adifferent cell type or revert to a less differentiated cell type.De-differentiation refers to the process by which a cell reverts to aless specialized (or committed) position within the lineage of a cell.As used herein, the lineage of a cell defines the heredity of the cell,i.e. which cells it came from and what cells it can give rise to. Thelineage of a cell places the cell within a hereditary scheme ofdevelopment and differentiation.

In a broad sense, a progenitor cell is a cell that has the capacity tocreate progeny that are more differentiated than itself, and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in greater detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a non-renewing progenitor cell or as anintermediate progenitor or precursor cell.

As used herein, the phrase differentiates into a mesodermal, ectodermalor endodermal lineage refers to a cell that becomes committed to aspecific mesodermal, ectodermal or endodermal lineage, respectively.Examples of cells that differentiate into a mesodermal lineage or giverise to specific mesodermal cells include, but are not limited to, cellsthat are adipogenic, chondrogenic, cardiogenic, dermatogenic,hematopoetic, hemangiogenic, myogenic, nephrogenic, urogenitogenic,osteogenic, pericardiogenic, or stromal. Examples of cells thatdifferentiate into ectodermal lineage include, but are not limited toepidermal cells, neurogenic cells, and neurogliagenic cells. Examples ofcells that differentiate into endodermal lineage include, but are notlimited to, pleurigenic cells, hepatogenic cells, cells that give riseto the lining of the intestine, and cells that give rise to pancreogenicand splanchogenic cells.

The cells of the present invention are generally referred to asumbilicus-derived cells (or UDCs). They also may sometimes be referredto more generally herein as postpartum-derived cells or postpartum cells(PPDCs). In addition, the cells may be described as being stem orprogenitor cells, the latter term being used in the broad sense. Theterm derived is used to indicate that the cells have been obtained fromtheir biological source and grown or otherwise manipulated in vitro(e.g., cultured in a growth medium to expand the population and/or toproduce a cell line). The in vitro manipulations of umbilical stem cellsand the unique features of the umbilicus-derived cells of the presentinvention are described in detail below.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition (“in culture” or “cultured”). A primary cellculture is a culture of cells, tissues, or organs taken directly from anorganism(s) before the first subculture. Cells are expanded in culturewhen they are placed in a growth medium under conditions that facilitatecell growth and/or division, resulting in a larger population of thecells. When cells are expanded in culture, the rate of cellproliferation is sometimes measured by the amount of time needed for thecells to double in number. This is referred to as doubling time.

A cell line is a population of cells formed by one or moresubcultivations of a primary cell culture. Each round of subculturing isreferred to as a passage. When cells are subcultured, they are referredto as having been passaged. A specific population of cells, or a cellline, is sometimes referred to or characterized by the number of timesit has been passaged. For example, a cultured cell population that hasbeen passaged ten times may be referred to as a P10 culture. The primaryculture, i.e., the first culture following the isolation of cells fromtissue, is designated P10. Following the first subculture, the cells aredescribed as a secondary culture (P1 or passage 1). After the secondsubculture, the cells become a tertiary culture (P2 or passage 2), andso on. It will be understood by those of skill in the art that there maybe many population doublings during the period of passaging; thereforethe number of population doublings of a culture is greater than thepassage number. The expansion of cells (i.e., the number of populationdoublings) during the period between passaging depends on many factors,including but not limited to the seeding density, substrate, medium,growth conditions, and time between passaging.

A conditioned medium is a medium in which a specific cell or populationof cells has been cultured, and then removed. When cells are cultured ina medium, they may secrete cellular factors that can provide trophicsupport to other cells. Such trophic factors include, but are notlimited to hormones, cytokines, extracellular matrix (ECM), proteins,vesicles, antibodies, and granules. The medium containing the cellularfactors is the conditioned medium.

Generally, a trophicfactor is defined as a substance that promotes or atleast supports, survival, growth, proliferation and/or maturation of acell, or stimulates increased activity of a cell.

When referring to cultured vertebrate cells, the term senescence (alsoreplicative senescence or cellular senescence) refers to a propertyattributable to finite cell cultures; namely, their inability to growbeyond a finite number of population doublings (sometimes referred to asHayflick's limit). Although cellular senescence was first describedusing fibroblast-like cells, most normal human cell types that can begrown successfully in culture undergo cellular senescence. The in vitrolifespan of different cell types varies, but the maximum lifespan istypically fewer than 100 population doublings (this is the number ofdoublings for all the cells in the culture to become senescent and thusrender the culture unable to divide). Senescence does not depend onchronological time, but rather is measured by the number of celldivisions, or population doublings, the culture has undergone. Thus,cells made quiescent by removing essential growth factors are able toresume growth and division when the growth factors are re-introduced,and thereafter carry out the same number of doublings as equivalentcells grown continuously. Similarly, when cells are frozen in liquidnitrogen after various numbers of population doublings and then thawedand cultured, they undergo substantially the same number of doublings ascells maintained unfrozen in culture. Senescent cells are not dead ordying cells; they are actually resistant to programmed cell death(apoptosis), and have been maintained in their nondividing state for aslong as three years. These cells are very much alive and metabolicallyactive, but they do not divide. The nondividing state of senescent cellshas not yet been found to be reversible by any biological, chemical, orviral agent.

As used herein, the term Growth Medium generally refers to a mediumsufficient for the culturing of umbilicus-derived cells. In particular,one presently preferred medium for the culturing of the cells of theinvention herein comprises Dulbecco's Modified Essential Media (alsoabbreviated DMEM herein). Particularly preferred is DMEM-low glucose(also DMEM-LG herein) (Invitrogen, Carlsbad, Calif.). The DMEM-lowglucose is preferably supplemented with 15% (v/v) fetal bovine serum(e.g. defined fetal bovine serum, Hyclone, Logan Utah),antibiotics/antimycotics (preferably penicillin (100 Units/milliliter),streptomycin (100 milligrams/milliliter), and amphotericin B (0.25micrograms/milliliter), (Invitrogen, Carlsbad, Calif.)), and 0.001%(v/v) 2-mercaptoethanol (Sigma, St. Louis Mo.). In some cases differentgrowth media are used, or different supplementations are provided, andthese are normally indicated in the text as supplementations to GrowthMedium.

Also relating to the present invention, the term standard growthconditions, as used herein refers to culturing of cells at 37° C., in astandard atmosphere comprising 5% CO₂. Relative humidity is maintainedat about 100%. While foregoing the conditions are useful for culturing,it is to be understood that such conditions are capable of being variedby the skilled artisan who will appreciate the options available in theart for culturing cells, for example, varying the temperature, CO₂,relative humidity, oxygen, growth medium, and the like.

The following abbreviations are used herein:

ANG2 (or Ang2) for angiopoietin 2;

APC for antigen-presenting cells;

BDNF for brain-derived neurotrophic factor;

bFGF for basic fibroblast growth factor;

bid (or BID) for “bis in die” (twice per day);

BSP for bone sialoprotein;

CK18 for cytokeratin 18;

CXC ligand 3 for chemokine receptor ligand 3;

DAPI for 4′-6-diamidino-2-phenylindole-2-HCl DMEM for Dulbecco's MinimalEssential Medium;

DMEM:lg (or DMEM:Lg, DMEM:LG) for DMEM with low glucose;

EDTA for ethylenediaminetetraacetic acid;

EGF for epidermal growth factor;

ERG FOR ELECTRORETMALGRAM

FACS for fluorescent activated cell sorting;

FBS for fetal bovine serum;

GCP-2 for granulocyte chemotactic protein 2;

GFAP for glial fibrillary acidic protein;

HB-EGF for heparin-binding epidermal growth factor;

HCAEC for human coronary artery endothelial cells;

HGF for hepatocyte growth factor;

hMSC for human mesenchymal stem cells;

HNF-1alpha for hepatocyte-specific transcription factor;

HUVEC for human umbilical vein endothelial cells;

1309 for a chemokine and the ligand for the CCR8 receptor and isresponsible for chemoattraction of TH2 type T-cells. I309 binds toendothelial cells, stimulates chemotaxis and invasion of these cells,and enhances HUVEC differentiation into capillary-like structures in anin vitro Matrigel assay. Furthermore, 1309 is an inducer of angiogenesisin vivo in both the rabbit cornea and the chick chorioallantoic membraneassay (CAM).

IL-6 for interleukin-6;

IL-8 for interleukin-8;

K19 for keratin 19;

K8 for keratin 8;

KGF for keratinocyte growth factor;

MCP-1 for monocyte chemotactic protein 1;

MDC for macrophage-derived chemokine;

MIP1alpha for macrophage inflammatory protein 1alpha;

MIP1beta for macrophage inflammatory protein 1beta;

MSC for mesenchymal stem cells;

NHDF for normal human dermal fibroblasts;

NPE for Neural Progenitor Expansion media;

PBMC for peripheral blood mononuclear cell;

PBS for phosphate buffered saline;

PDGFbb for platelet derived growth factor;

PDGFr/alpha for platelet derived growth factor receptor alpha;

PD-L2 for programmed-death ligand 2;

PE for phycoerythrin

PO for “per os” (by mouth);

PPDC for postpartum-derived cell;

Rantes (or RANTES) for regulated on activation, normal T cell expressedand secreted;

rhGDF-5 for recombinant human growth and differentiation factor 5;

SC for subcutaneously;

SDF-1alpha for stromal-derived factor 1alpha;

SHH for sonic hedgehog;

SOP for standard operating procedure;

TARC for thymus and activation-regulated chemokine;

TCP for Tissue culture plastic;

TGFbeta2 for transforming growth factor beta2;

TGFbeta-3 for transforming growth factor beta-3;

TIMP1 for tissue inhibitor of matrix metalloproteinase 1;

TPO for thrombopoietin;

TUJ1 for BIII Tubulin;

UDC for umbilicus-derived cell;

VEGF for vascular endothelial growth factor;

vWF for von Willebrand factor;

alphaFP for alpha-fetoprotein;

Description

Various patents and other publications are cited herein and throughoutthe specification, each of which is incorporated in its entirety byreference herein.

In a first aspect, the invention provides isolated umbilicus-derivedcells comprising cells derived from human umbilical cord tissuesubstantially free of blood. The cells are capable of self-renewal andexpansion in culture. The umbilicus-derived cells have the potential todifferentiate into cells of other phenotypes. In preferred embodiments,the cells can differentiate into any cell of ectodermal, mesodermal, orendodermal origin. The invention provides, in one of its several aspectscells that are isolated from human umbilical tissues. The cells, asdisclosed herein, preferably are not derived from umbilical cord blood.Nor are they endothelial cells derived from, for example, blood vessels.Rather, the cells are derived of the remaining umbilicus tissues.

The cells have been characterized as to several of their cellular,genetic, immunological, and biochemical properties. For example, thecells have been well-characterized by their cell surface markers, bytheir gene expression, by their ability to produce certain biochemicaltrophic factors, and by their immunological properties.

In another of its several aspects, cell cultures comprising the isolatedumbilicus-derived cells of the invention are provided. The cultures arefree of maternal cells in preferred embodiments herein. Also preferredare cells that have a normal karyotype, and those that maintain theirkaryotype as they are passaged. Most highly preferred are those cellsthat have and maintain a normal karyotype throughout passaging until atleast after senesecence.

Methods of culturing and expanding umbilicus-derived cells and cellcultures comprising them are provided. Presently preferred are cellsthat require no added growth factors, but rather are capable ofexpansion in many available culture media, especially those supplementedwith for example, fetal bovine serum. Preferred media for culturing andexpansion of the cells include Growth Medium, defined herein as mediumcomprising Dulbecco's Modified Essential Media (also abbreviated DMEMherein). Preferred is DMEM-low glucose (also DMEM-LG herein)(Invitrogen, Carlsbad, Calif.). The DMEM-low glucose is most preferablysupplemented with 15% (v/v) fetal bovine serum (e.g. defined fetalbovine serum, Hyclone, Logan Utah), antibiotics/antimycotics (preferablypenicillin at 100 Units/milliliter, streptomycin at 100micrograms/milliliter, and amphotericin B at 0.25 micrograms/milliliter;(Invitrogen, Carlsbad, Calif.)), and 0.001% (v/v) 2-mercaptoethanol(Sigma, St. Louis Mo.).

The skilled artisan will appreciate that the Growth Medium can bevariously supplemented and altered in any of the ways known in the art,and may be optimized for particular reasons. In addition, the cells areable to grow in many other culture media, including chemically definedmedia in the absence of added serum. Several such media are exemplifiedbelow. In addition to routine culturing and maintenance of the cells,many other media are known in the art for affecting differentiation ofsuch potent cells into specific types of cells or progenitors ofspecific cells. The skilled artisan will appreciate that these media areuseful for many purposes, and are included within the scope of theinvention, but they are not necessarily preferred for routine culturingand expansion.

In addition to the flexibility of the cells with respect to culturingmedium, the cells can grow under a variety of environmental conditions.In particular, the cells can grow under a wide range of atmosphericconditions. Presently preferred are atmospheres which range from about5% O₂ to about 20% or more O₂. The cells grow and expand well in GrowthMedium under these conditions, typically in the presence of about 5%CO₂, and the balance of the atmosphere as nitrogen. The skilled artisanwill appreciate that the cells may tolerate broader ranges of conditionsin different media, and that optimization for specific purposes may beappropriate.

Although the cells have not demonstrated any requirements for specificgrowth factors, the cells have demonstrated a requirement for L-valineover D-valine. Thus preferred culture media should have the L-isomer ofthis amino acid present. In addition to the demonstrated flexibility ofthe cells with respect to their growth requirements, preferred are theisolated cells that can attach and expand on either a coated or anuncoated tissue culture vessel. Tissue culture vessels include plates,flasks, tubes and the like, made of any of the materials known in theart—e.g. plastic, polystyrene, glass. Such vessels, where coated, arecoated with any of a variety of compounds as are known in the art.Presently preferred coated vessels comprise a coating with gelatin,laminin, collagen, polyornithine, polylysine, vitronectin, orfibronectin, for example.

The isolated cells of the invention also preferably expand in thepresence of from about 2% to about 15% serum, preferably Fetal BovineSerum, and more preferably defined Fetal Bovine Serum. The cells alsoexpand in the presence or absence of beta-mercaptoethanol, and in thepresence or absence of added growth factors including one or more ofEGF, bFGF, PDGF, VEGF, IGF-I, and LIF. The skilled artisan willappreciate that these flexible growth requirements allow for manyoptions when culturing or working with these cells. In certainembodiments, cells are grown in Advanced DMEM (Gibco) containing any oneof the previously mentioned growth factors, particularly bFGF. Asdescribed above, while presently Growth Medium is preferred, there is noabsolute requirement for serum; growth has been accomplished inserum-free medium.

In a preferred embodiment, the cells have excellent doubling andexpansion potential and are suitable for use in diagnostic andtherapeutic applications because their numbers can be scaled up readily.The isolated cells preferably can double sufficiently to generate yieldsof greater than about 10¹⁴ cells in less than about 80 days in culturewhen seeded at about 10³ cells/cm² in a suitable medium. More preferredare cells that can double sufficiently to generate greater than about10¹⁵ cells in less than about 80 days in culture when seeded at about5,000 cells/cm². Still more preferred is the isolated cell which candouble sufficiently to generate greater than about 10¹⁷ cells in lessthan about 65 days in culture when seeded at about 5,000 cells/cm².

The isolated cells of the invention can undergo extensive doublings.Preferably, the cells undergo at least 30 doublings before reachingsenescence. More preferably, at least 40 doublings in culture areattainable. Still more preferable are those cells that can achieve morethan 40 doublings before becoming senescent. The cells of the inventionare preferably able to undergo division over a longer period of time,than are, for example, human mesenchymal cells, or human fibroblasts,cultured under the same conditions.

In one embodiment, umbilical cells are isolated in the presence of oneor more enzyme activities. A broad range of digestive enzymes for use incell isolation from tissue are known in the art, including enzymesranging from those considered weakly digestive (e.g. deoxyribonucleasesand the neutral protease, dispase) to strongly digestive (e.g. papainand trypsin). Presently preferred are mucolytic enzyme activities,metalloproteases, neutral proteases, serine proteases (such as trypsin,chymotrypsin, or elastase), and deoxyribonucleases. More preferred areenzyme activites selected from metalloproteases, neutral proteases andmucolytic activities. Presently preferred are cells that are isolated inthe presence of one or more activities of collagenase, hyaluronidase anddispase. More preferred are those cells isolated in the presence of acollagenase from Clostridium histolyticum, and either of the proteaseactivities, dispase and thermolysin. Still more preferred are cellsisolated with collagenase and dispase enzyme activities. Also preferredare such cells isolated in the presence of a hyaluronidase activity, inaddition to collagenase and dispase activity.

In another aspect of the invention, isolated umbilicus-derived cellshaving specific cell surface marker expression profiles are provided.Preferred cells are characterized in either their production or lack ofproduction of one or more cell surface markers selected from CD10, CD13,CD31, CD44, CD45, CD73, CD90, CD117, CD141, PDGFr-alpha, HLA-A,B,C, andHL-DR, DP,DQ. More preferred are cells that are characterized withrespect to their production, or lack thereof, of several (e.g., two,four, five, or eight), or even ten of, or all of, the foregoing, toprovide a profile of the cell. In preferred embodiments, the cellsproduce one or more of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, orHLA-A,B,C. More preferred are cells that express several or all of theforegoing markers. Still more preferred are those cell which expresseach of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, and HLA-A,B,C.

Preferred cells may also be characterized with respect to markers theydo not produce, and such information is also useful in forming acharacterization or immunological profile of the cell. Preferably, thecells do not produce one or more of CD31, CD34, CD45, CD117, CD141, orHLA-DR,DP,DQ, as detected by flow cytometry. More preferred are cellsthat do not produce several or more of the foregoing. Still morepreferred are cells for which production of none of CD31, CD34, CD45,CD117, CD141, or LA-DR,DP,DQ can be detected by flow cytometry under theconditions described herein.

In other preferred embodiments, the cells can be shown to produceseveral or more of the CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, andHLA-A,B,C and concomitantly not produce one, or several, or more ofCD31, CD34, CD45, CD117, CD 141, or HLA-DR,DP,DQ as detected by flowcytometry. More highly preferred are cells that produce each of CD10,CD13, CD44, CD73, CD90, PDGFr-alpha, and HLA-A,B,C and concomitantly notproduce any of CD31, CD34, CD45, CD117, CD141, or HLA-DR,DP,DQ asdetected by flow cytometry.

Such cells are highly characterized with respect to their production ofthese cell surface proteins. In preferred embodiments, thecharacterization of the cells with repect to such production remainssubstantially constant and does not change substantially with variationsin isolation procedure, passage, culture conditions, or even the coatingor lack thereof on a tissure culture vessel.

The cells of the invention have also been characterized according totheir expression of a wide variety of genes. Accordingly, another aspectof the invention provides isolated human umbilicus-derived cells, whichare characterized, relative to a human cell that is a fibroblast, amesenchymal stem cell, or an ileac crest bone marrow cell, in theirexpression of genes for: short stature homeobox 2; heat shock 27 kDaprotein 2; chemokine (C-X-C motif) ligand 12 (stromal cell-derivedfactor 1); elastin (supravalvular aortic stenosis, Williams-Beurensyndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (from cloneDKFZp586M2022); mesenchyme homeobox 2 (growth arrest-specific homeobox);sine oculis homeobox homolog 1 (Drosophila); crystallin, alpha B;dishevelled associated activator of morphogenesis 2; DKFZP586B2420protein; similar to neuralin 1; tetranectin (plasminogen bindingprotein); src homology three (SH3) and cysteine rich domain; B-celltranslocation gene 1, anti-proliferative; cholesterol 25-hydroxylase;runt-related transcription factor 3; hypothetical protein FLJ23191;interleukin 11 receptor, alpha; procollagen C-endopeptidase enhancer;frizzled homolog 7 (Drosophila); hypothetical gene BC008967; collagen,type VIII, alpha 1; tenascin C (hexabrachion); iroquois homeobox protein5; hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2; Homosapiens cDNA FLJ12280 fis, clone MAMMA1001744; cytokine receptor-likefactor 1; potassium intermediate/small conductance calcium-activatedchannel, subfamily N, member 4; integrin, alpha 7; DKFZP586L151 protein;transcriptional co-activator with PDZ-binding motif (TAZ); sine oculishomeobox homolog 2 (Drosophila); KIAA1034 protein; early growth response3; distal-less homeobox 5; hypothetical protein FLJ20373; aldo-ketoreductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase,type II); biglycan; fibronectin 1; proenkephalin; integrin, beta-like 1(with EGF-like repeat domains); Homo sapiens mRNA full length insertcDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNADKFZp564B222 (from clone DKFZp564B222); vesicle-associated membraneprotein 5 (myobrevin); EGF-containing fibulin-like extracellular matrixprotein 1; BCL2/adenovirus EIB 19 kDa interacting protein 3-like; AEbinding protein 1; cytochrome c oxidase subunit VIIa polypeptide 1(muscle); neuroblastoma, suppression of tumorigenicity 1; insulin-likegrowth factor binding protein 2, 36 kDa; as well as interleukin 8;reticulon 1; chemokine (C-X-C motif) ligand 1 (melonoma growthstimulating activity, alpha); chemokine (C-X-C motif) ligand 6(granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3;and tumor necrosis factor, alpha-induced protein 3.

In one embodiment, preferred cells have the cell surface markercharacterization described above and are further characterized in theirrelative gene expression. For example, some preferred cells have thecell surface characterization described above and express a gene for oneor more of interleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1(melonoma growth stimulating activity, alpha); chemokine (C-X-C motif)ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-C motif)ligand 3; or tumor necrosis factor, alpha-induced protein 3. Morepreferred are those cells that express a gene for several, (e.g., atleast two, four, five or more) of each of the foregoing. The cells arecapable of self-renewal and expansion in culture, and have the potentialto differentiate into cells of other phenotypes. Also provided aretherapeutic cell cultures comprising the isolated humanumbilicus-derived cells.

Also provided are UDCs comprising the cell surface markercharacterization and which relative to a human cell that is afibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell,have reduced expression of one or more genes selected from: shortstature homeobox 2; heat shock 27 kDa protein 2; chemokine (C-X-C motif)ligand 12 (stromal cell-derived factor 1); elastin (supravalvular aorticstenosis, Williams-Beuren syndrome); Homo sapiens mRNA; cDNADKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeobox 2 (growtharrest-specific homeobox); sine oculis homeobox homolog 1 (Drosophila);crystallin, alpha B; dishevelled associated activator of morphogenesis2; DKFZP586B2420 protein; similar to neuralin 1; tetranectin(plasminogen binding protein); src homology three (SH3) and cysteinerich domain; B-cell translocation gene 1, anti-proliferative;cholesterol 25-hydroxylase; runt-related transcription factor 3;hypothetical protein FLJ23191; interleukin 11 receptor, alpha;procollagen C-endopeptidase enhancer; frizzled homolog 7 (Drosophila);hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C(hexabrachion); iroquois homeobox protein 5; hephaestin; integrin, beta8; synaptic vesicle glycoprotein 2; Homo sapiens cDNA FLJ12280 fis,clone MAMMA1001744; cytokine receptor-like factor 1; potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 4; integrin, alpha 7; DKFZP586L151 protein; transcriptionalco-activator with PDZ-binding motif (TAZ); sine oculis homeobox homolog2 (Drosophila); KIAA1034 protein; early growth response 3; distal-lesshomeobox 5; hypothetical protein FLJ20373; aldo-keto reductase family 1,member C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan;fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-likerepeat domains); Homo sapiens mRNA full length insert cDNA cloneEUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptorC/guanylate cyclase C (atrionatriuretic peptide receptor C);hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222(from clone DKFZp564B222); vesicle-associated membrane protein 5(myobrevin); EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; insulin-like growthfactor binding protein 2, 36 kDa. Preferred are cells that have reducedexpression of a gene for several (e.g., at least 5, 10, 15, 20, 25, 30,35, 40, 45, 50 or more, or even all) of the foregoing.

Still more highly preferred are those cells having the cell surfacemarker characterization as described herein above and which also expressa gene for each of interleukin 8; reticulon 1; chemokine (C-X-C motif)ligand 1 (melonoma growth stimulating activity, alpha); chemokine (C-X-Cmotif) ligand 6 (granulocyte); chemokine (C-X-C motif) ligand 3; ortumor necrosis factor, alpha-induced protein 3, and which relative to ahuman cell that is a fibroblast, a mesenchymal stem cell, or an ileaccrest bone marrow cell, have reduced expression of one or more genesselected from: short stature homeobox 2; heat shock 27 kDa protein 2;chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);elastin (supravalvular aortic stenosis, Williams-Beuren syndrome); Homosapiens mRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeobox 2 (growth arrest-specific homeobox); sine oculis homeoboxhomolog 1 (Drosophila); crystallin, alpha B; dishevelled associatedactivator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin1; tetranectin (plasminogen binding protein); src homology three (SH3)and cysteine rich domain; B-cell translocation gene 1,anti-proliferative; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; hypothetical protein FLJ23191; interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog7 (Drosophila); hypothetical gene BC008967; collagen, type VIII, alpha1; tenascin C (hexabrachion); iroquois homeobox protein 5; hephaestin;integrin, beta 8; synaptic vesicle glycoprotein 2; Homo sapiens cDNAFLJ12280 fis, clone MAMMA1001744; cytokine receptor-like factor 1;potassium intermediate/small conductance calcium-activated channel,subfamily N, member 4; integrin, alpha 7; DKFZP586L151 protein;transcriptional co-activator with PDZ-binding motif (TAZ); sine oculishomeobox homolog 2 (Drosophila); KIAA1034 protein; early growth response3; distal-less homeobox 5; hypothetical protein FLJ20373; aldo-ketoreductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase,type II); biglycan; fibronectin 1; proenkephalin; integrin, beta-like 1(with EGF-like repeat domains); Homo sapiens mRNA full length insertcDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNADKFZp564B222 (from clone DKFZp564B222); vesicle-associated membraneprotein 5 (myobrevin); EGF-containing fibulin-like extracellular matrixprotein 1; BCL2/adenovirus E1B19 kDa interacting protein 3-like; AEbinding protein 1; cytochrome c oxidase subunit VIIa polypeptide 1(muscle); neuroblastoma, suppression of tumorigenicity 1; insulin-likegrowth factor binding protein 2, 36 kDa.

In another of its aspects, the invention provides isolated humanumbilicus-derived cells, which relative to a human cell that is afibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell,have reduced expression of genes for each of: short stature homeobox 2;heat shock 27 kDa protein 2; chemokine (C-X-C motif) ligand 12 (stromalcell-derived factor 1); elastin (supravalvular aortic stenosis,Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (fromclone DKFZp586M2022); mesenchyme homeobox 2 (growth arrest-specifichomeobox); sine oculis homeobox homolog 1 (Drosophila); crystallin,alpha B; dishevelled associated activator of morphogenesis 2;DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogenbinding protein); src homology three (SH3) and cysteine rich domain;B-cell translocation gene 1, anti-proliferative; cholesterol25-hydroxylase; runt-related transcription factor 3; hypotheticalprotein FLJ23191; interleukin 11 receptor, alpha; procollagenC-endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypotheticalgene BC008967; collagen, type VII, alpha 1; tenascin C (hexabrachion);iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; Homo sapiens cDNA FLJ12280 fis, cloneMAMMA1001744; cytokine receptor-like factor 1; potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 4; integrin, alpha 7; DKFZP586L151 protein; transcriptionalco-activator with PDZ-binding motif (TAZ); sine oculis homeobox homolog2 (Drosophila); KIAA1034 protein; early growth response 3; distal-lesshomeobox 5; hypothetical protein FLJ20373; aldo-keto reductase family 1,member C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan;fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-likerepeat domains); Homo sapiens mRNA full length insert cDNA cloneEUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptorC/guanylate cyclase C (atrionatriuretic peptide receptor C);hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222(from clone DKFZp564B222); vesicle-associated membrane protein 5(myobrevin); EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; insulin-like growthfactor binding protein 2, 36 kDa; and which express a gene for each ofinterleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melonomagrowth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6(granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3;and tumor necrosis factor, alpha-induced protein 3, wherein theexpression is increased relative to that of a human cell which is afibroblast, a mesenchymal stem cell, an ileac crest bone marrow cell, orplacenta-derived cell. Such cells need not be characterized with respectto their cell surface markers, but such cells preferably are capable ofself-renewal and expansion in culture and also have the ability todifferentiate in culture. Most preferred are cells which are isolated asadherent cells as described below, however, such cells can also be grownafter isolation in a spherical form under certain conditions, thus theyare not obligatorily adherent.

Certain preferred embodiments include cells as described above whichalso produce vimentin or alpha-smooth muscle actin. More preferred arecells that produce both vimentin and alpha-smooth muscle actin. Theproduction of these proteins appears to distinguish the cells of theinstant invention from hematopoietic cells isolated form umbical cordblood for example.

In preferred embodiments, the above gene expression profiles aresubstantially stable and do not vary with passaging or normal culturingconditions. Of course, it is understood that such a profile may varywhen cells are grown under conditions which stimulate or inducedifferentiation into other phenotypes, for example, or the expression ofa different set of genes.

The invention also provides therapeutic cell cultures comprising thecell having the cell surface production or gene expression profiles, orboth, as described above. Cell banks comprising therapeutic cultures aresimilarly included with in the scope of the invention. For example, acell bank may include cultured cells of the invention at variouspassages, as well as cells of the invention which have been induced todifferentiate to different phenotypes. Other cells may be successfullybanked either separately or in co-culture with the cells of theinvention. A complete cell bank may include the banked cells of a widevariety of individuals. In preferred embodiments, banked cells arestored cryopreserved at −180° C. for example. Cells are preferablystored at −90° C. in some embodiments. The cells of the invention arereadily cryopreserved under a variety of conditions such as are known inthe art.

In preferred embodiments, the cells lack the cell surface moleculesrequired to substantially stimulate allogeneic lymphocytes in a mixedlymphocyte reaction. In more preferred embodiments, the cells lack thesurface molecules required to substantially stimulate CD4⁺T-cells in invitro assessments, or in vivo in allogeneic, syngeneic, or autologousrecipients. Still more preferred are those cells that do cause not anysubstantial adverse immunological consequences for in vivo applications.The therapeutic cell cultures lack detectable amounts of at least two,or several, or all of the stimulating proteins HLA-DR, HLA-DP, HLA-DQ,CD80, CD86, and B7-H2, as determined by flow cytometry. Those lackingall of the foregoing are most preferred. Also preferred are therapeuticcell cultures which further lack detectable amounts of one or both ofthe immuno-modulating proteins HLA-G and CD178, as determined by flowcytometry. Also preferred are therapeutic cell cultures which expressdetectable amounts of the immuno-modulating protein PD-L2, as determinedby flow cytometry. In one embodiment, the therapeutic cell culture doesnot substantially stimulate a lymphocyte mediated response in vitro, ascompared to allogeneic controls in a mixed lymphocyte reaction.

In another of its several aspects, the invention provides isolated humanumbilicus-derived cells capable of self-renewal and expansion in cultureand which have the potential to differentiate into cells of otherphenotypes, wherein the cells do not substantially stimulate allogeneiclymphocytes in a mixed lymphocyte reaction. More preferred are cellswhich do not substantially stimulate CD4⁺T cells, and which producePD-L2, but not one or more of HLA-G, CD178, HLA-DR, HLA-DP, HLA-DQ,CD80, CD86, or B7-H2. The cells also can produce vimentin oralpha-smooth muscle actin in certain embodiments. More preferred arecells producing both vimentin and alpha-smooth muscle actin. Still morepreferred are those cells not producing any of HLA-G, CD178, HLA-DR,HLA-DP, HLA-DQ, CD80, CD86, and B7-H2.

Cells of the invention which secrete useful molecules, for example,growth factors, are presently preferred. Such cells have utlity not onlyfor the their cellular properties but for their secreted molecules, forexample in conditioned medium, or cell-free lysates. In another aspect,the invention provides isolated human umbilicus-derived cells thatsecrete one or more of the angiogenic factors MCP-1, IL-8, GCP-2, HGF,KGF, FGF, HB-EGF, TPO, or TIMP1. In certain embodiments, the cellssecrete all of the foregoing factors. The cells do not secrete one ormore of the angiogenic factors SDF-1 alpha, TGF-beta2, ANG2, PDGFbb orVEGF, as detected by ELISA. In certain embodiments they secrete none ofSDF-1alpha, TGF-beta2, ANG2, PDGFbb or VEGF, as detected by ELISA.

In another aspect, the cells of the invention are defined according to acombination of many of the characteristics provided herein. For example,the invention provides isolated umbilicus-derived cells comprisingL-valine-requiring cells derived from mammalian postpartum tissuesubstantially free of blood. The cells are capable of self-renewal andexpansion in culture and have the potential to differentiate into cellsof other phenotypes; for example cardiomyocytes, or their progenitors.Cells may be isolated from umbilicus tissue of any mammal of interest bythe techniques provided herein. Human cells are presently preferred. Thecells can be grown under a wide range of conditions, including a widevariety of culture media, and environmental conditions. The cells can begrown at least from about 35° C. to about 39° C., and possibly a widerrange depending on other conditions. The cells can be grown inchemically-defined media, or in medium with added mammalian serum, forexample fetal bovine serum. The cells also tolerate cryopreservation atvarious stages. Cells can be maintained frozen, or banked attemperatures preferably at or below −80° C. for long periods. Otherpreferred temperatures range from about −90° C. to about −180° C. orbelow. Specialized electric freezers can be used, or cells can be storedin the liquid or vapor phases of nitrogen. Tissues can also be bankedprior to the isolation of the cells. Preferably Good Banking Procedures,such as are known in the art, are followed.

The cells are capable of growth in atmospheres containing oxygen fromabout 5% to at least about 20% and comprise at least one of thefollowing characteristics: the cells have the potential for at leastabout 40 doublings in culture; the cells preferably are adherent, thusattachment and expansion on a coated or uncoated tissue culture vesselis preferred, wherein a coated tissue culture vessel comprises a coatingof gelatin, laminin, collagen, polyornithine, polylysine, vitronectin,or fibronectin;

The cells preferably produce of at least one of tissue factor, vimentin,and alpha-smooth muscle actin; more preferred are cells which produceeach of tissue factor, vimentin, and alpha-smooth muscle actin;production of at least one of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha,PD-L2 and HLA-A,B,C is also preferred. The cells are also characterizedin their lack of production of at least one of CD31, CD34, CD45, CD80,CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ, as detectedby flow cytometry; more preferable cells lack production of all of thesesurface markers. Also preferred are cells which express at least one ofinterleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melanomagrowth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6(granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3;and tumor necrosis factor, alpha-induced protein 3. Preferred cells alsohave expression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an ileac crest bone marrow cell, is reducedfor at least one of: short stature homeobox 2; heat shock 27 kDa protein2; chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);elastin (supravalvular aortic stenosis, Williams-Beuren syndrome); Homosapiens mRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeobox 2 (growth arrest-specific homeobox); sine oculis homeoboxhomolog 1 (Drosophila); crystallin, alpha B; dishevelled associatedactivator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin1; tetranectin (plasminogen binding protein); src homology three (SH3)and cysteine rich domain; B-cell translocation gene 1,anti-proliferative; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; hypothetical protein FLJ23191; interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog7 (Drosophila); hypothetical gene BC008967; collagen, type VIII, alpha1; tenascin C (hexabrachion); iroquois homeobox protein 5; hephaestin;integrin, beta 8; synaptic vesicle glycoprotein 2; Homo sapiens cDNAFLJ12280 fis, clone MAMMA1001744; cytokine receptor-like factor 1;potassium intermediate/small conductance calcium-activated channel,subfamily N, member 4; integrin, alpha 7; DKFZP586L151 protein;transcriptional co-activator with PDZ-binding motif (TAZ); sine oculishomeobox homolog 2 (Drosophila); KIAA1034 protein; early growth response3; distal-less homeobox 5; hypothetical protein FLJ20373; aldo-ketoreductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase,type II); biglycan; fibronectin 1; proenkephalin; integrin, beta-like 1(with EGF-like repeat domains); Homo sapiens mRNA full length insertcDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNADKFZp564B222 (from clone DKFZp564B222); vesicle-associated membraneprotein 5 (myobrevin); EGF-containing fibulin-like extracellular matrixprotein 1; BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AEbinding protein 1; cytochrome c oxidase subunit VIIa polypeptide 1(muscle); neuroblastoma, suppression of tumorigenicity 1; insulin-likegrowth factor binding protein 2, 36 kDa; the skilled artisan willappreciate that the expression of a wide variety of genes isconveniently characterized on a gene array, for example on a AffymetrixGENECHIP®.

The cells secrete a variety of biochemically active factors, such asgrowth factors, chemokines, cytokines and the like. Preferred cellssecrete at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF,BDNF, TPO, MIP1alpha, RANTES, and TIMP1; preferred cells mayalternatively be characterized in their lack of secretion of at leastone of TGF-beta2, ANG2, PDGFbb, MIP1beta, 1309, MDC, and VEGF, asdetected by ELISA. These and other characteristics are available toidentify and characterize the cells, and distinguish the cells of theinvention from others known in the art.

In preferred embodiments, the cell comprises two or more of theforegoing characteristics. More preferred are those cells comprising,three, four, or five or more of the characteristics. Still morepreferred are those isolated postpartum cells comprising six, seven, oreight or more of the characteristics. Still more preferred presently arethose cells comprising all nine of the claimed characteristics.

Also presently preferred are cells that produce at least two of tissuefactor, vimentin, and alpha-smooth muscle actin. More preferred arethose cells producing all three of the proteins tissue factor, vimentin,and alpha-smooth muscle actin.

The skilled artisan will appreciate that cell markers are subject tovary somewhat under vastly different growth conditions, and thatgenerally herein described are characterizations in Growth Medium, orvariations thereof. Postpartum-derived cells that produce of at leastone, two, three, or four of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha,PD-L2 and HLA-A,B,C are preferred. More preferred are those cellsproducing five, six, or seven of these cell surface markers. Still morepreferred are postpartum cells that can produce all eight of theforegoing cell surface marker proteins.

Similarly, postpartum cells that lack production of at least one, two,three, or four of the proteins CD31, CD34, CD45, CD80, CD86, CD117,CD141, CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ, as detected by flowcytometry are presently preferred. Cells lacking production of at leastfive, six, seven or eight or more of these markers are also preferred.More preferred are cells which lack production of at least nine or tenof the cell surface markers. Most highly preferred are those cellslacking production of all eleven of the foregoing identifying proteins.

Presently preferred cells produce each of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, and HLA-A,B,C, and do not produce any of CD31, CD34, CD45,CD117, CD141, or HLA-DR,DP,DQ, as detected by flow cytometry.

Presently, it is preferred that postpartum-derived cells express atleast one, two or three of interleukin 8; reticulon 1; chemokine (C-X-Cmotif) ligand 1 (melanoma growth stimulating activity, alpha); chemokine(C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine(C-X-C motif) ligand 3; and tumor necrosis factor, alpha-induced protein3. More preferred are those cells which express four or five, and stillmore preferred are cell capable of expressing all six of the foregoinggenes.

For some embodiments, preferred are cells, which relative to a humancell that is a fibroblast, a mesenchymal stem cell, or an ileac crestbone marrow cell, have reduced expression for at least one of the genescorresponding to: short stature homeobox 2; heat shock 27 kDa protein 2;chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);elastin (supravalvular aortic stenosis, Williams-Beuren syndrome); Homosapiens mRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeobox 2 (growth arrest-specific homeobox); sine oculis homeoboxhomolog 1 (Drosophila); crystallin, alpha B; dishevelled associatedactivator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin1; tetranectin (plasminogen binding protein); src homology three (SH3)and cysteine rich domain; B-cell translocation gene 1,anti-proliferative; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; hypothetical protein FLJ23191; interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog7 (Drosophila); hypothetical gene BC008967; collagen, type VIII, alpha1; tenascin C (hexabrachion); iroquois homeobox protein 5; hephaestin;integrin, beta 8; synaptic vesicle glycoprotein 2; Homo sapiens cDNAFLJ12280 fis, clone MAMMA1001744; cytokine receptor-like factor 1;potassium intermediate/small conductance calcium-activated channel,subfamily N, member 4; integrin, alpha 7; DKFZP586L151 protein;transcriptional co-activator with PDZ-binding motif (TAZ); sine oculishomeobox homolog 2 (Drosophila); KIAA1034 protein; early growth response3; distal-less homeobox 5; hypothetical protein FLJ20373; aldo-ketoreductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase,type II); biglycan; fibronectin 1; proenkephalin; integrin, beta-like 1(with EGF-like repeat domains); Homo sapiens mRNA full length insertcDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNADKFZp564B222 (from clone DKFZp564B222); vesicle-associated membraneprotein 5 (myobrevin); EGF-containing fibulin-like extracellular matrixprotein 1; BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AEbinding protein 1; cytochrome c oxidase subunit VIIa polypeptide 1(muscle); neuroblastoma, suppression of tumorigenicity 1; insulin-likegrowth factor binding protein 2, 36 kDa. More preferred are cells thathave, relative to human fibroblasts, mesenchymal stem cells, or ileaccrest bone marrow cells, reduced expression of at least 5, 10, 15 or 20genes corresponding to those listed above. Presently more preferred arecell with reduced expression of at least 25, 30, or 35 of the genescorresponding to the listed sequences. Also more preferred are thosepostpartum-derived cells having expression that is reduced, relative tothat of a human fibroblast, a mesenchymal stem cell, or an ileac crestbone marrow cell, of genes corresponding to 35 or more, 40 or more, oreven all of the sequences listed.

Secretion of certain growth factors and other cellular proteins can makecells of the invention particularly useful. Preferred postpartum-derivedcells secrete at least one, two, three or four of MCP-1, IL-6, IL-8,GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1. Cellswhich secrete more than five, six, seven or eight of the listed proteinsare also useful and preferred. Cells which can secrete at least nine,ten, eleven or more of the factors are more preferred, as are cellswhich can secrete twelve or more, or even all thirteen of the proteinsin the foregoing list.

While secretion of such factors is useful, cells can also becharacterized by their lack of secretion of factors into the medium.Umbilicus-derived cells that lack secretion of at least one, two, threeor four of TGF-beta2, ANG2, PDGFbb, MIP1beta, 1309, MDC, and VEGF, asdetected by ELISA, are presently preferred for use. Cells that arecharacterized in their lack of secretion of five or six of the foregoingproteins are more preferred. Cells which lack secretion of all seven ofthe factors listed above are also preferred.

In another aspect of the invention, therapeutic cell cultures areprovided, the cell cultures comprising the isolated cells as describedabove for use in treating patients in need of angiogenesis-stimulatingtrophic factors. Such therapeutic cell cultures are also provided foruse in treating a patient in need of neural growth stimulating trophicfactors.

Methods of deriving UDCs from human umbilical tissue are provided. Thecells are capable of self-renewal and expansion in culture, and have thepotential to differentiate into cells of other phenotypes. The methodcomprises (a) obtaining human umbilical tissue; (b) removingsubstantially all of blood to yield a substantially blood-free umbilicaltissue, (c) dissociating the tissue by mechanical or enzymatictreatment, or both, (d) resuspending the tissue in a culture medium, and(e) providing growth conditions which allow for the growth of a humanumbilicus-derived cell capable of self-renewal and expansion in cultureand having the potential to differentiate into cells of otherphenotypes.

Tissue can be obtained from any completed pregnancy, term or less thanterm, whether delivered vaginally, or through other routes, for examplesurgical Cesarean section. Obtaining tissue from tissue banks is alsoconsidered within the scope of the present invention.

The tissue is rendered substantially free of blood by any means known inthe art. For example, the blood can be physically removed by washing,rinsing, and diluting and the like, before or after bulk blood removalfor example by suctioning or draining. Other means of obtaining a tissuesubstantially free of blood cells might include enzymatic or chemicaltreatment.

Dissociation of the umbilical tissues can be accomplished by any of thevarious techniques known in the art, including by mechanical disruption,for example, tissue can be aseptically cut with scissors, or a scalpel,or such tissue can be otherwise minced, blended, ground, or homogenizedin any manner that is compatible with recovering intact or viable cellsfrom human tissue.

In a presently preferred embodiment, the isolation procedure alsoutilizes an enzymatic digestion process. Many enzymes are known in theart to be useful for the isolation of individual cells from complextissue matrices to facilitate growth in culture. As discussed above, abroad range of digestive enzymes for use in cell isolation from tissueis available to the skilled artisan. Ranging from weakly digestive (e.g.deoxyribonucleases and the neutral protease, dispase) to stronglydigestive (e.g. papain and trypsin), such enzymes are availablecommercially. A nonexhaustive list of enzymes compatable herewithincludes mucolytic enzyme activities, metalloproteases, neutralproteases, serine proteases (such as trypsin, chymotrypsin, orelastase), and deoxyribonucleases. Presently preferred are enzymeactivites selected from metalloproteases, neutral proteases andmucolytic activities. For example, collagenases are known to be usefulfor isolating various cells from tissues. Deoxyribonucleases can digestsingle-stranded DNA and can minimize cell-clumping during isolation.Enzymes can be used alone or in combination. Serine protease arepreferably used in a sequence following the use of other enzymes as theymay degrade the other enzymes being used. The temperature and time ofcontact with serine proteases must be monitored. Serine proteases may beinhibited with alpha 2 microglobulin in serum and therefore the mediumused for digestion is preferably serum-free. EDTA and DNase are commonlyused and may improve yields or efficiencies. Preferred methods involveenzymatic treatment with for example collagenase and dispase, orcollagenase, dispase, and hyaluronidase, and such methods are providedwherein in certain preferred embodiments, a mixture of collagenase andthe neutral protease dispase are used in the dissociating step. Morepreferred are those methods which employ digestion in the presence of atleast one collagenase from Clostridium histolyticum, and either of theprotease activities, dispase and thermolysin. Still more preferred aremethods employing digestion with both collagenase and dispase enzymeactivities. Also preferred are methods which include digestion with ahyaluronidase activity in addition to collagenase and dispaseactivities. The skilled artisan will appreciate that many such enzymetreatments are known in the art for isolating cells from various tissuesources. For example, the LIBERASE Blendzyme (Roche) series of enzymecombinations are very useful and may be used in the instant methods.Other sources of enzymes are known, and the skilled artisan may alsoobtain such enzymes directly from their natural sources. The skilledartisan is also well-equipped to assess new, or additional enzymes orenzyme combinations for their utility in isolating the cells of theinvention. Preferred enzyme treatments are 0.5, 1, 1.5, or 2 hours longor longer. In other preferred embodiments, the tissue is incubated at37° C. during the enzyme treatment of the dissociation step. Dilutingthe digest may also improve yields of cells as cells may be trappedwithin a viscous digest.

While the use of enzyme activites is presently preferred, it is notrequired for isolation methods as provided herein. Methods based onmechanical separation alone may be successful in isolating the instantcells from the umbilicus as discussed above.

The cells can be resuspended after the tissue is dissociated into anyculture medium as discussed herein above. Cells may be resuspendedfollowing a centrifugation step to separate out the cells from tissue orother debris. Resuspension may involve mechanical methods ofresuspending, or simply the addition of culture medium to the cells.

Providing the growth conditions allows for a wide range of options as toculture medium, supplements, atmospheric conditions, and relativehumidity for the cells. A preferred temperature is 37° C., however thetemperature may range from about 35° C. to 39° C. depending on the otherculture conditions and desired use of the cells or culture.

Presently preferred are methods which provide cells which require noexogenous growth factors, except as are available in the supplementalserum provided with the Growth Medium. Also provided herein are methodsof deriving umbilical cells capable of expansion in the absence ofparticular growth factors. The methods are similar to the method above,however they require that the particular growth factors (for which thecells have no requirement) be absent in the culture medium in which thecells are ultimately resuspended and grown in. In this sense, the methodis selective for those cells capable of division in the absence of theparticular growth factors. Preferred cells in some embodiments arecapable of growth and expansion in chemically-defined growth media withno serum added. In such cases, the cells may require certain growthfactors, which can be added to the medium to support and sustain thecells. Presently preferred factors to be added for growth on serum-freemedia include one or more of FGF, EGF, IGF, and PDGF. In more preferredembodiments, two, three or all four of the factors are add to serum freeor chemically defined media. In other embodiments, LIF is added toserum-free medium to support or improve growth of the cells.

Also provided are methods wherein the cells can expand in the presenceof from about 5% to about 20% oxygen in their atmosphere. Methods toobtain cells that require L-valine require that cells be cultured in thepresence of L-valine. After a cell is obtained, its need for L-valinecan be tested and confirmed by growing on D-valine containing mediumthat lacks the L-isomer.

Methods are provided wherein the cells can undergo at least 25, 30, 35,or 40 doublings prior to reaching a senescent state. Methods forderiving cells capable of doubling to reach 10¹⁴ cells or more areprovided. Preferred are those methods which derive cells that can doublesufficiently to produce at least about 10¹⁴, 10¹⁵, 10¹⁶, or 10¹⁷ or morecells when seeded at from about 10³ to about 10⁶ cells/cm² in culture.Preferably these cell numbers are produced within 80, 70, or 60 days orless.

Isolated human umbilicus-derived cells derived by the above method arealso provided herein. The cells maintain a consistent normal karyotypenotwithstanding repeated passaging in certain embodiments. Also providedare cultures of human umbilicus-derived cells derived by the abovemethod, wherein the cultures are free of maternal cells. Therapeuticcultures are provided which are obtained by the methods of theinvention.

There are many uses for the cells of the instant invention. For example,because of the trophic factors secreted by the cells, they may be usefulfor the growth, maintenance, support or the like, of other useful cells,either directly or indirectly. For example, the cells may be used tomake conditioned media, or may be cocultured with another cells ofinterest, including direct or indirect ex vivo coculture of cellsintended to be used therapeutically in an autologous recipient. Fordirect coculture, the cells may be mixed together, while for indirectcoculture, the cells may be cultured in compartments separated bysemi-permeable membranes which exclude cell migration. For example,polycarbonate, polyester (PET), and collagen-coatedpolytetrafluoroethylene (PTFE) membranes of about less 3 microns nominalpore size exclude cell migration and are useful for such purposes. Suchmembranes for culture systems are able to exchange, for example, solublegrowth factors between the umbilical-derived cells and another celltype. Examples are known in the art and some are available commerically(e.g. TRANSWELL culture membranes (Corning Inc, Corning N.Y.)).

Also provided herein are therapeutic compositions comprising anumbilicus-derived cell and another therapeutic agent or factor. Suchfactors include, but are not limited to, IGF, LIF, PDGF, EGF, FGF, aswell as antithrombogenic and antiapoptotic agents. Such therapeuticcompositions can further comprise one or more additional cell types.

In addition to the above, compositions derived from the cells themselvesare provided herein. Cell lysates, soluble cell fractions andmembrane-enriched cell fractions are all provided herein. Cell lysatesinclude lysates in which substantially all, and more preferably all ofthe cells have been lysed, for example by mechanical shear, enzymatictreatment, detergent or other chemical treatment, and the like, orcombinations thereof. The resulting lysates include all of thecomponents of the cell and have many utilities, for example insupporting or maintaining growth of the cells of the invention or othercells of interest. In addition the cell lysates can serve as startingmaterial for the purification or enrichment of desirable cell products.In preferred embodiments, cell lysates are further separated into atleast a soluble cell fraction and a membrane-enriched cell fraction.Each may be useful for specific purposes. For example, where in vivouses are contemplated, for example administration by injection, solublecell fractions may be particularly useful in minimizing stimulation ofallogeneic PBMCs. More particularly, soluble cell fractions preferablylack the required antigens to stimulate adverse immunological reactions,for example stimulation of allogeneic lymphocytes, allogeneic CD4⁺ Tcells, or even naïve CD4⁺ T cells. Extracellular matrices derived fromthe cells, for example comprising basement membranes are also useful andare provided herein. Such extracellular material is useful for in vitroassays, for example, angiogenesis assays. They also may be useful invivo as part of a therapeutic regimen. For example, extracellular matrixcompounds are known for use in augmentation and repair such asapplications relating to healing of acute and chronic wounds, or incosmetic and reconstructive surgery. The extracellular matrix materialis often useful where collagen-like properties are desirable.Extracellular matrix material is particularly useful in applicationrequiring elasticity or viscosity because the extracellular matrixappears to be able to provide these attributes. Another application ofextracellular matrix is in combination with an implantable therapeuticdevice. Techniques for cellular disruption range from gentle to harsh,the skilled artisan is well-equipped to select the technique for celldisruption based on the end-use of the cell lystate so obtained.

Compositions of the invention also include conditioned culture media asprovided herein. Such media have first been used to grow the cells orcultures of the invention, which during growth secrete one or moreuseful products into the medium. Conditioned medium from these novelcells are useful for many purposes, including for example, supportingthe growth of other mammalian cells in need of growth factors or trophicfactors secreted into the media by the cells and cultures of theinvention, and promoting, for example, angiogenesis. Conditioned mediaare useful for the secreted growth factors they contain, and preferablythe conditioned media of the instant invention contain one or moregrowth factors useful for supporting the growth of another mammaliancell in need of such growth factors. The use of conditioned media iswell understood by those of skill in the art. Continuous culture systemsfor generating conditioned culture medium are contemplated herein, andthe cells of the invention are useful for large scale or continuousproduction of secreted cellular products such as growth factors.

Preferably, the conditioned media of the invention comprise one or moreof MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, or TIMP1.More preferred are those conditioned media which comprise several,(e.g., at least two, three or four) of the foregoing. Still morepreferred are those which contain many (e.g., at least five, six, seveneight or more) of the growth factors listed. Also preferred areconditioned media which comprise additional secreted molecules ofinterest.

Another embodiment provides a mammalian cell culture comprising theconditioned medium and a mammalian cell in need of MCP-1, IL-6, IL-8,GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, or TIMP1. Culturing inconditioned media provides for indirect methods of supporting, forexample, the growth or maintenance of other cells. For example, suchindirect methods provide ways of expanding populations of mammaliancells, which due to their growth factor requirements, are difficult toexpand in culture in the absence of the conditioned medium.

Another aspect of the invention provides co-cultures comprising theumbilicus-derived cells and another mammalian cell of any phenotype.These co-cultures can comprise cells of any phenotype and may, forexample, provide a way of expanding populations of mammalian cells,which due to their growth factor requirements are difficult to expand.This is a way of using the properties of the umbilicus-derived cellsdirectly to support the growth of other cells. Such co-cultures are alsoof use in vivo for various applications, particularly where the use ofsuch cells does not generate an undesired immunological response. Thepresence of the umbilicus-derived cells can encourage establishment andgrowth of cells, for example into implanted scaffolds, or at surgicalrepair sites. In preferred embodiments, the co-cultures comprise a humancell line in addition to the umbilicus-derived cell. Still morepreferred would be to use the cells in a family member or person closelyrelated to the donor. More preferred yet would be the co-cultures foruse in the individual from whom the cells were derived.

In other aspects of the invention, uses of the cultures are provided.For example, the cultures of umbilicus-derived cells are useful inpromoting angiogenesis. They are also useful in the treatment of softtissue disease or injury. In preferred embodiments, the therapeutic cellcultures are grown on a scaffold or matrix. The growth on the scaffoldor matrix or similar substrate can be in vivo, in vitro, or acombination of these. For in vivo application, the substrate can besurgically applied, for example as a repair to damage soft tissue. Thesubstrate can be seeded with UDCs, or pretreated with UDC-conditionedmedium, or cell lysates or soluble cell extracts, or other cellfraction, including an extracellular matrix, prior to surgical use. Forin vitro applications, the cells can help other cells to establish on orwithin the substrate or its interstices. For example, skin or other softtissue of dermal or subdermal layers can be grown in vitro on suchsubstrates for later use. For combination uses, a population of cellscan be established in vitro on the substrate or scaffold and thensurgically attached, grafted, or implanted for further growth.Preestablishing a cell population, particularly one that secretesangiogenic factors may lead to much faster healing. Presently preferredfor such scaffolds are nonpermanent, bioabsorbable polymeric matricesand the like.

A variety of scaffolds or matrices are known in the arts of tissueengineering and wound healing for example, and are useful with the cellsand methods herein. Examples include, but are not limited to mats,foams, or self assembling peptides. Preferred mats comprise nonwovenfibers. Nonwoven mats may, for example, be formed using fibers comprisedof a synthetic absorbable copolymer of glycolic and lactic acids(PGA/PLA), sold under the tradename VICRYL (Ethicon, Inc., Somerville,N.J.). Foams preferred for use herein include porous foams such as thosecomprising, for example, poly(epsilon-caprolactone)/poly(glycolic acid)(PCL/PGA) copolymer, as discussed in U.S. Pat. No. 6,355,699. Selfassembling peptides and hydrogels formed therefrom are known in the art,and include RAD 16 hydrogel, exemplified herein. Such materials arefrequently used as supports for growth of cells or tissue.

Also provided herein are additional uses for the therapeutic cellcultures of the invention that include, but are not limited to, use inthe treatment of bone disease or injury, the treatment of pancreaticdisease or injury, the treatment of kidney disease or injury, thetreatment of neural disease or injury, cardiac disease or injury, andthe treatment of hepatic disease or injury.

In another of its aspects, the invention provides higher orderstructures such as implantable tissue structures comprising the cells ofthe invention, implantable human tissue matrix comprising the cells,human tissues comprising the cells, and human organs comprising thesecells.

The invention also provides, in another of its several aspects,injectable therapeutic cells comprising isolated human umbilicus-derivedcells capable of self-renewal and expansion in culture and having thepotential to differentiate into cells of other phenotypes, wherein thecells do not substantially stimulate allogeneic lymphocytes in a mixedlymphocyte reaction, and produce PD-L2, but not HLA-G, CD178, HLA-DR,HLA-DP, HLA-DQ, CD80, CD86, or B7-H2. This injectable therapeutic cellcomposition is useful as a therapeutic treatment in any applicationwhere undifferentiated cells can be recruited to a site through thecirculation. In preferred embodiments, the injectable cell is treated toinactivate tissue factor. Such treatment includes any treatment capableof inactivating tissue factor without destroying the integrity of thecell. More preferred are treatments which have no effect on viability,doubling time or expansion. The injectable cell is treated with ananti-tissue factor antibody in one presently preferred embodiment.

In conjunction with therapeutic cells, other biologically activemolecules, such as antithrombogenic agents, anti-apoptotic agents, andanti-inflammatory agents may be useful and may be administered insequence with, or coadministered with the the cells, individually or incombinations or two or more such compounds or agents. For example,anti-apoptotic agents may be useful to minimize programmed cell death.Such agents include but are not limited to EPO, EPO derivatives andanalogs, and their salts, TPO, IGF-I, IGF-II, hepatocyte growth factor(HGF), and caspase inhibitors. Anti-inflammatory agents include but arenot limited to P38 MAP kinase inhibitors, statins, IL-6 and IL-1inhibitors, Pemirolast, Tranilast, Remicade, Sirolimus, nonsteroidalanti-inflammatory compounds, for example, Tepoxalin, Tolmetin, andSuprofen.

Other bioactive factors or therapeutic agents which can becoadministered with the therapeutic cells of the invention include, forexample, antithrombogenic factors, immunosuppressive or immunomodulatoryagents, and antioxidants. Examples of immunosuppressive and immudulatoryagents include calcineurin inhibitors, for example cyclosporine,Tacrolimus, mTOR inhibitors such as Sirolimus or Everolimus;anti-proliferatives such as azathioprine and mycophenolate mofetil;corticosteroids for example prednisolone or hydrocortisone; antibodiessuch as monoclonal anti-IL-2Rα receptor antibodies, Basiliximab,Daclizumab; polyclonal anti-T-cell antibodies such as anti-thymocyteglobulin (ATG), anti-lymphocyte globulin (ALG), and the monoclonalanti-T cell antibody OKT3. Antithrombogenic compounds which can betherapeutically provided in conjunction with the cells of the inventioninclude, for example, heparin, heparin derivatives, urokinase, and PPack(dextrophenylalanine proline arginine chloromethylketone); antithrombincompounds, platelet receptor antagonists, anti-thrombin antibodies,anti-platelet receptor antibodies, aspirin, dipyridamole, protamine,hirudin, prostaglandin inhibitors, and platelet inhibitors. Antioxidantsare well known in the art of course and any pharamacueitcally acceptableantioxidant may be administered in conjunction with the cells of theinvention including probucol; vitamins A, C, and E, coenzyme Q-10,glutathione, L cysteine, N-acetylcysteine, or antioxidant derivative,analogs or salts of the foregoing.

In another aspect of the invention, kits for the growth and maintenance,the isolation and the use of the umbilical-derived cells are provided.The cells, cell lysates, soluble cell fractions, membrane fractions andmatrices can conveniently be employed as parts of kits, for example, fora kit for culture or implantation. The invention provides a kitincluding the UDCs and additional components, including instructions forgrowth or maintenance, isolation, or use of the cells or cell fractions,together with for example, matrix (e.g., a scaffold) material, hydratingagents (e.g., physiologically-compatible saline solutions, prepared cellculture media), cell culture substrates (e.g., culture dishes, plates,vials, etc.), cell culture media (whether in liquid or dehydrated form),antibiotic compounds, hormones, and the like. Kits for growth can forexample include all of the components of the Growth Medium as usedherein, including serum, for example fetal bovine serum. While the kitcan include any such components, preferably it includes all ingredientsnecessary for its intended use. If desired, the kit also can includecells (typically cryopreserved), which can be seeded into the lattice asdescribed herein. Kits for isolation will contain everything required topractice the isolation methods as provided herein, except for theumbilicus tissue which should be obtained fresh or frozen from a tissuebank at the time of isolation. The surgical equipment for dissociatingthe tissue, preferred enzymes, or choices of enzymes in stable form areprovided, as are the buffers and medium, cell strainers and the like, asrequired or preferred for the method as disclosed above. Detailedinstructions with optional steps and lists of suppliers of optional oralternative materials are alos conveniently provided. Control cells canbe included for comparison of the cells isolated to, for example the UDCcultures deposited with the ATCC. Kits for utilizing theumbilicus-derived cells preferably contain populations of the cells, ortherapeutic compositions comprising the cells, components and products,or fractions or conditioned media derived from the cells as describedabove. In some embodiments, the kits may include one or more cellpopulations, including at least UDCs and a pharmaceutically acceptablecarrier (liquid, semi-solid or solid). The populations in someembodiments are homogenous or even clonal cell lines of UDCs. In otherembodiments, the kits include other cell lines for use in coculture.Therapeutic application kits preferably include additional bioactiveagents as desired for example anithrombogenic agents, anti-inflammatoryagents, antiapoptotic agents, and immunosuppressive or immunomodulatorycompounds. The kits also optionally may include a means of administeringthe cells, for example by injection. The kits further may includeinstructions for use of the cells. Kits prepared for field hospital use,such as for military use, may include full-procedure supplies includingtissue scaffolds, surgical sutures, and the like, where the cells are tobe used in conjunction with repair of acute injuries. Kits for assaysand in vitro methods as described herein may contain one or more of (1)UDCs or fractions, components or products of UDCs, (2) reagents forpracticing the in vitro method, (3) other cells or cell populations, asappropriate, for example for cocultures and (4) instructions forconducting the in vitro method.

The following examples describe several aspects of embodiments of theinvention in greater detail. These examples are intended to furtherillustrate aspects of the invention described herein. These examplesshould not be construed to limit the aspect so exemplified.

EXAMPLE 1 Isolation of Cells from Postpartum Umbilicus Tissues

Postpartum umbilicus tissues were obtained upon birth of either a fullterm or pre-term pregnancy. Cells were harvested from five separatedonors of umbilicus tissue. Different methods of cell isolation weretested for their ability to yield cells with: 1) the potential todifferentiate into cells with different phenotypes, a characteristiccommon to stem cells, or 2) the potential to provide critical trophicfactors useful for other cells and tissues.

Methods & Materials

Umbilical cell isolation. Umbilical cords were obtained from NationalDisease Research Interchange (NDRI, Philadelphia, Pa.). The tissues wereobtained following normal deliveries. The cell isolation protocols wereperformed aseptically in a laminar flow hood. To remove blood anddebris, the cord was washed in phosphate buffered saline (PBS;Invitrogen, Carlsbad, Calif.) in the presence of penicillin at 100Units/milliliter and streptomycin at 100 milligrams/milliliter, andamphotericin B at 0.25 micrograms/milliliter (Invitrogen Carlsbad,Calif.). The tissues were then mechanically dissociated in 150 cm²tissue culture plates in the presence of 50 milliliters of medium(DMEM-Low glucose or DMEM-High glucose; Invitrogen), until the tissuewas minced into a fine pulp. The chopped tissues were transferred to 50milliliter conical tubes (approximately 5 grams of tissue per tube).

The tissue was then digested in either DMEM-Low glucose medium orDMEM-High glucose medium, each containing 100 Units/milliliter,streptomycin at 100 milligrams/milliliter, and amphotericin B at 0.25micrograms/milliliter and the digestion enzymes. In some experiments anenzyme mixture of collagenase and dispase was used (“C:D”) (collagenase(Sigma, St Louis, Mo.), 500 Units/milliliter; and dispase (Invitrogen),50 Units/milliliter, in DMEM-Low glucose medium). In other experiments amixture of collagenase, dispase and hyaluronidase (“C:D:H”) was used(C:D:H=collagenase, 500 Units/milliliter; dispase, 50 Units/milliliter;and hyaluronidase (Sigma), 5 Units/milliliter, in DMEM-Low glucose). Theconical tubes containing the tissue, medium and digestion enzymes wereincubated at 37° C. in an orbital shaker (Environ, Brooklyn, N.Y.) at225 rpm for 2 hrs.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,the supernatant was aspirated. The pellet was resuspended in 20milliliters of Growth Medium (DMEM:Low glucose (Invitrogen), 15 percent(v/v) fetal bovine serum (FBS; defined fetal bovine serum; Lot#AND18475; Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol(Sigma), penicillin at 100 Units per milliliter, streptomycin at 100micrograms per milliliter, and amphotericin B at 0.25 micrograms permilliliter; (each from Invitrogen, Carlsbad, Calif.)). The cellsuspension was filtered through a 70-micron nylon BD FALCON CellStrainer (BD Biosciences, San Jose, Calif.). An additional 5 millilitersrinse comprising Growth Medium was passed through the strainer. The cellsuspension was then passed through a 40-micrometer nylon cell strainer(BD Biosciences, San Jose, Calif.) and chased with a rinse of anadditional 5 milliliters of Growth Medium.

The filtrate was resuspended in Growth Medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cells were resuspended in 50 milliliters of freshgrowth medium. This process was repeated twice more.

After the final centrifugation, supernatant was aspirated and the cellpellet was resuspended in 5 milliliters of fresh growth medium. Thenumber of viable cells was determined using trypan blue staining. Cellswere then cultured under standard conditions.

The cells isolated from umbilical cord tissues were seeded at 5,000cells/cm² onto gelatin-coated T-75 flasks (Corning Inc., Corning, N.Y.)in Growth Medium. After two days, spent medium and unadhered cells wereaspirated from the flasks. Adherent cells were washed with PBS threetimes to remove debris and blood-derived cells. Cells were thenreplenished with Growth Medium and allowed to grow to confluence (about10 days from passage 0) to passage 1. On subsequent passages (frompassage 1 to 2 etc), cells reached sub-confluence (75-85 percentconfluence) in 4-5 days. For these subsequent passages, cells wereseeded at 5,000 cells/cm². Cells were grown in a humidified incubatorwith 5 percent carbon dioxide at 37° C.

Cell Isolation using LIBERASE Blendzymes. Cells were isolated frompostpartum tissues in DMEM-Low glucose medium with LIBERASE (2.5milligrams per milliliter, Blendzyme 3; Roche Applied Sciences,Indianapolis, Ind.) and hyaluronidase (5 Units/milliliter, Sigma).Digestion of the tissue and isolation of the cells was as described forother protease digestions above, however, the LIBERASE/hyaluronidasemixture was used instead of the C:D or C:D:H enzyme mixture. Tissuedigestion with LIBERASE resulted in the isolation of cell populationsfrom postpartum tissues that expanded readily.

Cell isolation using other enzyme combinations. Procedures were comparedfor isolating cells from the umbilical cord using differing enzymecombinations. Enzymes compared for digestion included: i) collagenase;ii) dispase; iii) hyaluronidase; iv) collagenase:dispase mixture (C:D);v) collagenase:hyaluronidase mixture (C:H); vi) dispase:hyaluronidasemixture (D:H); and vii) collagenase:dispase:hyaluronidase mixture(C:D:H). Differences in cell isolation utilizing these different enzymedigestion conditions were observed (Table 1-1).

Isolation of cells from residual blood in the cords. Other attempts weremade to isolate pools of cells from umbilical cord by differentapproaches. In one instance umbilical cord was sliced and washed withgrowth medium to dislodge the blood clots and gelatinous material. Themixture of blood, gelatinous material and growth medium was collectedand centrifuged at 150×g. The pellet was resuspended and seeded ontogelatin coated flasks in growth medium. From these experiments a cellpopulation was isolated that readily expanded.

Isolation of cells from cord blood. Cells have also been isolated fromcord blood samples attained from NDRI. The isolation protocol used wasthat of International Patent Application US0229971 by Ho et al. Samples(50 milliliter and 10.5 milliliters, respectively) of umbilical cordblood (NDRI, Philadelphia Pa.) were mixed with lysis buffer(filter-sterilized 155 millimolar ammonium chloride, 10 millimolarpotassium bicarbonate, 0.1 millimolar EDTA buffered to pH 7.2 (allcomponents from Sigma, St. Louis, Mo.)). Cells were lysed at a ratio of1:20 cord blood to lysis buffer. The resulting cell suspension wasvortexed for 5 seconds, and incubated for 2 minutes at ambienttemperature. The lysate was centrifuged (10 minutes at 200×g). The cellpellet was resuspended in Complete Minimal Essential Medium (Gibco,Carlsbad Calif.) containing 10 percent fetal bovine serum (Hyclone,Logan Utah), 4 millimolar glutamine (Mediatech Herndon, Va.), penicillinat 100 Units per milliliter and streptomycin at 100 micrograms permilliliter (Gibco, Carlsbad, Calif.). The resuspended cells werecentrifuged (10 minutes at 200×g), the supernatant was aspirated, andthe cell pellet was washed in complete medium. Cells were seededdirectly into either T75 flasks (Corning, N.Y.), T75 laminin-coatedflasks, or T175 fibronectin-coated flasks (both Becton Dickinson,Bedford, Mass.).

Isolation of cells using different enzyme combinations and growthconditions. To determine whether cell populations could be isolatedunder different conditions and expanded under a variety of conditionsimmediately after isolation, cells were digested in Growth Medium withor without 0.001 percent (v/v) 2-mercaptoethanol (Sigma, St. Louis,Mo.), using the enzyme combination of C:D:H, according to the proceduresprovided above. All cells were grown in the presence of penicillin at100 Units per milliliter and streptomycin at 100 micrograms permilliliter. Under all tested conditions cells attached and expanded wellbetween passage 0 and 1 (Table 1-2). Cells in conditions 5-8 and 13-16were demonstrated to proliferate well up to 4 passages after seeding atwhich point they were cryopreserved.

Results

Cell isolation using different enzyme combinations. The combination ofC:D:H, provided the best cell yield following isolation, and generatedcells that expanded for many more generations in culture than the otherconditions (Table 1-1). An expandable cell population was not attainedusing collagenase or hyaluronidase alone. No attempt was made todetermine if this result is specific to the collagenase that was tested.TABLE 1-1 Isolation of cells from umbilical cord tissue using varyingenzyme combinations Enzyme Digest Cells Isolated Cell ExpansionCollagenase X X Dispase + (>10 h) + Hyaluronidase X XCollagenase:Dispase ++ (<3 h) ++ Collagenase:Hyaluronidase ++ (<3 h) +Dispase:Hyaluronidase + (>10 h) + Collagenase:Dispase:Hyaluronidase +++(<3 h) +++Key:+ = good,++ = very good,+++ = excellent,X = no success

Isolation of cells using different enzyme combinations and growthconditions. Cells attached and expanded well between passage 0 and 1under all conditions tested for enzyme digestion and growth (Table 1-2).Cells in experimental conditions 5-8 and 13-16 proliferated well up to 4passages after seeding, at which point they were cryopreserved. Allcells were cryopreserved for further analysis. TABLE 1-2 Isolation andculture expansion of postpartum cells under varying conditions:Condition Medium 15% FBS BME Gelatin 20% O₂ Growth Factors 1 DMEM-Lg Y YY Y N 2 DMEM-Lg Y Y Y N (5%) N 3 DMEM-Lg Y Y N Y N 4 DMEM-Lg Y Y N N(5%) N 5 DMEM-Lg N (2%) Y N (Laminin) Y EGF/FGF (20 ng/ml) 6 DMEM-Lg N(2%) Y N (Laminin) N (5%) EGF/FGF (20 ng/ml) 7 DMEM-Lg N (2%) Y N YPDGF/VEGF (Fibronectin) 8 DMEM-Lg N (2%) Y N N (5%) PDGF/VEGF(Fibronectin) 9 DMEM-Lg Y N Y Y N 10 DMEM-Lg Y N Y N (5%) N 11 DMEM-Lg YN N Y N 12 DMEM-Lg Y N N N (5%) N 13 DMEM-Lg N (2%) N N (Laminin) YEGF/FGF (20 ng/ml) 14 DMEM-Lg N (2%) N N (Laminin) N (5%) EGF/FGF (20ng/ml) 15 DMEM-Lg N (2%) N N Y PDGF/VEGF (Fibronectin) 16 DMEM-Lg N (2%)N N N (5%) PDGF/VEGF (Fibronectin)

Isolation of cells from residual blood in the cords. Nucleated cellsattached and grew rapidly. These cells were analyzed by flow cytometryand were similar to cells obtained by enzyme digestion.

Isolation of cells from cord blood. The preparations contained red bloodcells and platelets. No nucleated cells attached and divided during thefirst 3 weeks. The medium was changed 3 weeks after seeding and no cellswere observed to attach and grow.

Summary. Populations of cells could be isolated from umbilical tissueefficiently using the enzyme combination collagenase (ametalloprotease), dispase (neutral protease) and hyaluronidase(mucolytic enzyme which breaks down hyaluronic acid). LIBERASE, which isa blend of collagenase and a neutral protease, may also be used.Blendzyme 3, which is collagenase (4 Wunsch units/gram) and thermolysin(1714 casein Units/gram), was also used together with hyaluronidase toisolate cells. These cells expanded readily over many passages whencultured in growth expansion medium on gelatin coated plastic.

Cells were also isolated from residual blood in the cords, but not cordblood. The presence of cells in blood clots washed from the tissue,which adhere and grow under the conditions used, may be due to cellsbeing released during the dissection process.

REFERENCE

-   1. Ho, Tony, W., et al., WO2003025149 A2 “CELL POPULATIONS WHICH    CO-EXPRESS CD49C AND CD90” NEURONYX, INC. Application No. US0229971    US, Filed 20020920, A2 Published 20030327, A3 Published 20031218

EXAMPLE 2 Growth Characteristics of Umbilicus-Derived Cells

The cell expansion potential of umbilicus-derived cells was compared toother populations of isolated stem cells. The process of cell expansionto senescence is referred to as Hayflick's limit (Hayflick L. Thelongevity of cultured human cells. J. Am. Geriatr. Soc. 22(1):1-12,1974; Hayflick L. The strategy of senescence. Gerontologist14(1):37-45), 1974).

Materials and Methods

Gelatin-coating flasks. Tissue culture plastic flasks were coated byadding 20 milliliters 2% (w/v) gelatin (Type B: 225 Bloom; Sigma, StLouis, Mo.) to a T75 flask (Corning Inc., Corning, N.Y.) for 20 minutesat room temperature. After removing the gelatin solution, 10 millilitersphosphate-buffered saline (PBS) (Invitrogen, Carlsbad, Calif.) wereadded and then aspirated.

Comparison of expansion potential of umbilicus-derived cells with othercell populations. For comparison of growth expansion potential thefollowing cell populations were utilized; i) Mesenchymal stem cells(MSC; Cambrex, Walkersville, Md.); ii) Adipose-derived cells (U.S. Pat.No. 6,555,374 B1; U.S. Patent Application US20040058412); iii) Normaldermal skin fibroblasts (cc-2509 lot # 9F0844; Cambrex, Walkersville,Md.); and iv) Umbilicus-derived cells. Cells were initially seeded at5,000 cells/cm² on gelatin-coated T75 flasks in Growth Medium. Forsubsequent passages, cell cultures were treated as follows. Aftertrypsinization, viable cells were counted after trypan blue staining.Cell suspension (50 microliters) was combined with trypan blue (50microliters, Sigma, St. Louis Mo.). Viable cell numbers were estimatedusing a hemocytometer.

Following counting, cells were seeded at 5,000 cells/cm² ontogelatin-coated T 75 flasks in 25 milliliters of fresh Growth Medium.Cells were grown in a standard atmosphere (5 percent carbon dioxide(v/v)) at 37° C. The Growth Medium was changed twice per week. Whencells reached about 85 percent confluence they were passaged; thisprocess was repeated until the cells reached senescence.

At each passage, cells were trypsinized and counted. The viable cellyield, population doublings [ln(cells final/cells initial)/ln2], anddoubling time (time in culture/population doubling) were calculated. Forthe purposes of determining optimal cell expansion, the total cell yieldper passage was determined by multiplying the total yield for theprevious passage by the expansion factor for each passage (i.e.expansion factor=cells final/cells initial).

Expansion potential of cell banks at low density. The expansionpotential of cells banked at passage 10 was also tested. A different setof conditions was used. Normal dermal skin fibroblasts (cc-2509 lot #9F0844; Cambrex, Walkersville, Md.), umbilicus-derived cells, andplacenta-derived cells were tested. These cell populations had beenbanked at passage 10 previously, having been cultured at 5,000 cells/cm²at each passage to that point. The effect of cell density on the cellpopulations following cell thaw at passage 10 was determined. Cells werethawed under standard conditions, counted using trypan blue staining.Thawed cells were then seeded at 1,000 cells/cm² in Growth Medium. Cellswere grown under standard atmospheric conditions at 37° C. Growth Mediumwas changed twice a week. Cells were passaged as they reached about 85%confluence. Cells were subsequently passaged until senescence, i.e.,until they could not be expanded any further. Cells were trypsinized andcounted at each passage. The cell yield, population doubling (ln(cellsfinal/cells initial)/ln2) and doubling time (time in culture)/populationdoubling). The total cell yield per passage was determined bymultiplying total yield for the previous passage by the expansion factorfor each passage (i.e., expansion factor=cells final/cells initial).

Expansion of umbilicus-derived cells at low density from initial cellseeding. The expansion potential of freshly isolated umbilicus-derivedcell cultures under low cell seeding conditions was tested in anotherexperiment. Umbilicus-derived cells were isolated as described in aprevious example. Cells were seeded at 1,000 cells/cm and passaged asdescribed above until senescence. Cells were grown under standardatmospheric conditions at 37° C. Growth Medium was changed twice perweek. Cells were passaged as they reached about 85% confluence. At eachpassage, cells were trypsinized and counted by trypan blue staining. Thecell yield, population doubling (In (cell final/cell initial)/ln 2) anddoubling time (time in culture/population doubling) were calculated foreach passage. The total cell yield per passage was determined bymultiplying the total yield for the previous passage by the expansionfactor for each passage (i.e. expansion factor=cell final/cell initial).Cells were grown on gelatin and non-gelatin coated flasks.

Expansion of cells in low oxygen culture conditions. It has beendemonstrated that low O₂ cell culture conditions can improve cellexpansion in certain circumstances (Csete, Marie; Doyle, John; Wold,Barbara J.; McKay, Ron; Studer, Lorenz. Low oxygen culturing of centralnervous system progenitor cells. US20040005704). In order to determineif cell expansion of umbilicus-derived cells could be improved byaltering cell culture conditions, cultures of umbilicus-derived cellswere grown in low oxygen conditions. Cells were seeded at 5,000cells/cm² in Growth Medium on gelatin coated flasks. Cells wereinitially cultured under standard atmospheric conditions through passage5, at which point they were transferred to low oxygen (5% O₂) cultureconditions.

Other growth conditions. In other experiments cells were expanded onnon-coated, collagen-coated, fibronectin-coated, laminin-coated andmatrigel-coated plates. Cultures have been demonstrated to expand wellon these different matrices.

Results

Comparison of expansion potential of umbilicus-derived cells with othercell populations. Umbilicus-derived cells expanded for more than 40passages generating cell yields of >1E17 cells in 60 days. In contrast,MSCs and fibroblasts senesced after <25 days and <60 days, respectively.Although both adipose-derived and omental cells expanded for almost 60days they generated total cell yields of 4.5E12 and 4.24E13respectively. Thus, when seeded at 5,000 cells/cm² under theexperimental conditions utilized, umbilicus-derived cells expanded muchbetter than the other cell types grown under the same conditions (Table2-1). TABLE 2-1 Growth characteristics for different cell populationsgrown to senescence Total Population Yield Cell Type SenescenceDoublings (Total Cells) MSC 24 d 8 4.72E7  Adipose- 57 d 24  4.5E12derived cell Fibroblasts 53 d 26 2.82E13 Umbilical 65 d 42 6.15E17

Expansion of potential of cell banks at low density. Umbilicus-derivedand fibroblast cells expanded for greater than 10 passages generatingcell yields of >1E11 cells in 60 days (Table 2-2). Under theseconditions both the fibroblasts and the umbilicus-derived cellpopulations senesced after 80 days, completing >50 and >40 populationdoublings respectively. TABLE 2-2 Growth characteristics for differentcell populations using low density growth expansion from passage 10through senescence Cell Type Total Population Yield (Passage No.)Senescence Doublings (Total Cells) Fibroblast (P10) 80 days 43.682.59E11 Umbilical (P10) 80 days 53.6 1.25E14

Expansion of cells in low oxygen culture conditions. Cells expanded wellunder the reduced oxygen conditions, however, culturing under low oxygenconditions does not appear to have a significant effect on cellexpansion for postpartum-derived cells. These results are preliminary inthe sense that any ultimate conclusions to be made regarding the effectof reduced oxygen would best be drawn from experiments on growing cellsin low oxygen from initial isolation. Standard atmospheric conditionshave already proven successful for growing sufficient numbers of cells,and low oxygen culture is not required for the growth ofpostpartum-derived cells.

Summary. The current cell expansion conditions of growing isolatedumbilicus-derived cells at densities of about 5,000 cells/cm², in GrowthMedium on gelatin-coated or uncoated flasks, under standard atmosphericoxygen, are sufficient to generate large numbers of cells at passage 11.Furthermore, the data suggests that the cells can be readily expandedusing lower density culture conditions (e.g. 1,000 cells/cm²).Umbilicus-derived cell expansion in low oxygen conditions alsofacilitates cell expansion, although no incremental improvement in cellexpansion potential has yet been observed when utilizing theseconditions for growth. Presently, culturing umbilicus-derived cellsunder standard atmospheric conditions is preferred for generating largepools of cells. However, when the culture conditions are altered,umbilicus-derived cell expansion can likewise be altered. This strategymay be used to enhance the proliferative and differentiative capacity ofthese cell populations.

Under the conditions utilized, while the expansion potential of MSC andadipose-derived cells is limited, umbilicus-derived cells expand readilyto large numbers.

REFERENCES

-   1) Hayflick L. The longevity of cultured human cells. J Am Geriatr    Soc. 22:1-12, 1974.-   2) Hayflick L. The strategy of senescence. Gerontologist    14(1):37-45, 1974.-   3) United States Patent Application No. 20040058412-   4) United States Patent Application No. 20040048372-   6) Csete, Marie; Doyle, John; Wold, Barbara J.; McKay, Ron; and    Studer, Lorenz. Low oxygen culturing of central nervous system    progenitor cells. United States Patent Application No. 20040005704.

EXAMPLE 3 Growth of Umbilicus-Derived Cells in Medium ContainingD-Valine

It has been reported that medium containing D-valine instead of thenormal L-valine isoform can be used to selectively inhibit the growth offibroblast-like cells in culture (Hongpaisan, 2000; Sordillo et al.,1988). Experiments were performed to determine whether umbilicus-derivedcells could grow in medium containing D-valine.

Methods & Materials

Umbilicus-derived cells (P5) and fibroblasts (P9) were seeded at 5,000cells/cm² in gelatin-coated T75 flasks (Corning, Corning, N.Y.). After24 hours the medium was removed and the cells were washed with phosphatebuffered saline (PBS) (Gibco, Carlsbad, Calif.) to remove residualmedium. The medium was replaced with a modified Growth Medium (DMEM withD-valine (special order Gibco), 15% (v/v) dialyzed fetal bovine serum(Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma),penicillin at 50 Units/milliliter and streptomycin at 50milligrams/milliliter (Gibco)).

Results

Neither the umbilicus-derived cells nor the fibroblast cells seeded inthe D-valine-containing medium proliferated, unlike cells seeded inGrowth Medium containing dialyzed serum. Fibroblasts cells changedmorphologically, increasing in size and changing shape. All of the cellsdied and eventually detached from the flask surface after four weeks.Thus, it may be concluded that umbilicus-derived cells require L-valinefor cell growth and to maintain long-term viability.

REFERENCES

-   Hongpaisan J. (2000) Inhibition of proliferation of contaminating    fibroblasts by D-valine in cultures of smooth muscle cells from    human myometrium. Cell Biol Int. 24:1-7.-   Sordillo L M, Oliver S P, Akers R M. (1988) Culture of bovine    mammary epithelial cells in D-valine modified medium: selective    removal of contaminating fibroblasts. Cell Biol Int Rep. 12:355-64.

EXAMPLE 4 Karyotype Analysis of Umbilicus-Derived PPDCs

Cell lines used in cell therapy are preferably homogeneous and free fromany contaminating cell type. Human cells used in cell therapy shouldhave a normal number (46) of chromosomes with normal structure. Toidentify umbilicus-derived cell lines that are homogeneous and free fromcells of non-umbilical tissue origin, karyotypes of cell samples wereanalyzed.

Materials and Methods

PPDCs from postpartum tissue of a male neonate were cultured in GrowthMedia. Postpartum tissue from a male neonate (X,Y) was selected to allowdistinction between neonatal-derived cells and maternal derived cells(X,X). Cells were seeded at 5,000 cells per square centimeter in GrowthMedium in a T25 flask (Corning, Corning, N.Y.) and expanded to 80%confluence. A T25 flask containing cells was filled to the neck withGrowth Media. Samples were delivered to a clinical cytogenetics lab bycourier (estimated lab to lab transport time is one hour). Chromosomeanalysis was performed by the Center for Human & Molecular Genetics atthe New Jersey Medical School, Newark, N.J. Cells were analyzed duringmetaphase when the chromosomes are best visualized. Of twenty cells inmetaphase counted, five were analyzed for normal homogeneous karyotypenumber (two). A cell sample was characterized as homogeneous if twokaryotypes were observed. A cell sample was characterized asheterogeneous if more than two karyotypes were observed. Additionalmetaphase cells were counted and analyzed when a heterogeneous karyotypenumber (four) was identified.

Results

All cell samples sent for chromosome analysis were interpreted by asexhibiting a normal appearance. Each of the cell samples wascharacterized as homogeneous. (Table 4-1). TABLE 4-1 Karyotype resultsof PPDCs. Metaphase cells Metaphase Number of ISCN Tissue Passagecounted cells analyzed karyotypes Karyotype Umbilical 23 20 5 2 46, XXUmbilical 6 20 5 2 46, XY Umbilical 3 20 5 2 46, XX

Summary. Chromosome analysis identified umbilicus-derived PPDCs whosekaryotypes appear normal as interpreted by a clinical cytogeneticlaboratory. Karyotype analysis also identified cell lines free frommaternal cells, as determined by homogeneous karyotype.

EXAMPLE 5 Flow Cytometric Evaluation of Human Umbilicus-Derived CellSurface Markers

Characterization of cell surface proteins or “markers” by flow cytometrycan be used to determine a cell line's identity. The consistency ofexpression can be determined from multiple donors, and in cells exposedto different processing and culturing conditions. Postpartum cell linesisolated from the umbilicus were characterized by flow cytometry,providing a profile for the identification of these cell lines.

Materials and Methods

Media and Culture Vessels. Cells were cultured in Growth Medium, inplasma-treated 175, T150, and T225 tissue culture flasks (Corning,Corning, N.Y.) until confluent. The growth surfaces of the flasks werecoated with gelatin by incubating 2% (w/v) gelatin (Sigma, St. Louis,Mo.) for 20 minutes at room temperature.

Antibody Staining. Adherent cells in flasks were washed in phosphatebuffered saline (PBS); (Gibco, Carlsbad, Mo.) and detached withTrypsin/EDTA (Gibco). Cells were harvested, centrifuged, and resuspendedin 3% (v/v) FBS in PBS at a cell concentration of 1×10⁷ per milliliter.In accordance with the manufacture's specifications, antibody to thecell surface marker of interest (see below) was added to 100 microlitersof cell suspension and the mixture was incubated in the dark for 30minutes at 4° C. After incubation, cells were washed with PBS andcentrifuged to remove unbound antibody. Cells were resuspended in 500microliters PBS and analyzed by flow cytometry.

Flow Cytometry Analysis. Flow cytometry analysis was performed with aFACScalibur instrument (Becton Dickinson, San Jose, Calif.).

Antibodies to Cell Surface Markers. The following antibodies to cellsurface markers were used. TABLE 5-1 Antibodies used in characterizingcell surface markers of UDCs. Antibody Number Manufacture Catalog CD10BD Pharmingen (San Diego, CA) 555375 CD13 BD Pharmingen 555394 CD31 BDPharmingen 555446 CD34 BD Pharmingen 555821 CD44 BD Pharmingen 555478CD45RA BD Pharmingen 555489 CD73 BD Pharmingen 550257 CD90 BD Pharmingen555596 CD117 BD Pharmingen 340529 CD141 BD Pharmingen 559781 PDGFr-alphaBD Pharmingen 556002 HLA-A, B, C BD Pharmingen 555553 HLA-DR, DP, DQ BDPharmingen 555558 IgG-FITC Sigma (St. Louis, MO) F-6522 IgG-PE SigmaP-4685

Passage to Passage Comparison. Umbilicus-derived cells were analyzed atpassages 8, 15, and 20.

Donor to Donor Comparison. To compare differences among donors,umbilical from different donors were compared to each other.

Surface Coating Comparison. Umbilicus-derived cells cultured ongelatin-coated flasks was compared to umbilicus cultured on uncoatedflasks.

Results

Umbilicus-Derived Cell Characterization. Umbilicus-derived cellsanalyzed by flow cytometry showed positive expression of CD10, CD13,CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, indicated by theincreased values of fluorescence relative to the IgG control (data notshown). These cells were negative for detectable expression of CD31,CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, indicated by fluorescencevalues comparable to the IgG control (data not shown). Variations inflorescence values of positive curves were accounted for. The mean (i.e.CD13) and range (i.e. CD90) of the positive curves showed somevariation, but the curves appear normal, confirming a homogenouspopulation. Both curves individually exhibited values greater than theIgG control.

Passage to Passage Comparison. Umbilical cells at passage 8, 15, and 20analyzed by flow cytometry all expressed CD10, CD13, CD44, CD73, CD90,PDGFr-alpha and HLA-A, B, C, indicated by increased fluorescencerelative to the IgG control. These cells were negative for CD31, CD34,CD45, CD117, CD141, and HLA-DR, DP, DQ, indicated by fluorescence valuesconsistent with the IgG control. Variations in florescence detectionvalues of positive curves were within expected ranges. While the means(i.e. CD13) of the positive curves varied all curves individuallyexhibited values greater than the IgG control.

Donor to Donor Comparison. Umbilicus-derived cells isolated fromseparate donors analyzed by flow cytometry each showed positiveexpression of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C,reflected in the increased values of fluorescence relative to the IgGcontrol. These cells were negative for expression of CD31, CD34, CD45,CD117, CD141, and HLA-DR, DP, DQ with fluorescence values consistentwith the IgG control. Variations in florescence detection values ofpositive curves were accounted for. While the mean (i.e. CD10) of thepositive curves varied, both curves individually exhibited valuesgreater than the IgG control.

The Effect of Surface Coating with Gelatin. Umbilical cells expanded ongelatin and uncoated flasks analyzed by flow cytometry all were positivefor expression of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A,B, C, with increased values of fluorescence relative to the IgG control.These cells were negative for expression of CD31, CD34, CD45, CD117,CD141, and HLA-DR, DP, DQ, with fluorescence values consistent with theIgG control.

Summary. Analysis of umbilicus-derived postpartum cells by flowcytometry has established a profile useful to identify these cell lines.Umbilicus-derived postpartum cells are positive for CD10, CD13, CD44,CD73, CD90, PDGFr-alpha, HLA-A,B,C and negative for CD31, CD34, CD45,CD117, CD141 and HLA-DR, DP, DQ. This identity was consistent betweenvariations in variables including the donor, passage, culture vesselsurface coating, and digestion enzymes used in isolation and preparationof the cells. Some variation in individual fluorescence value histogramcurve means and ranges were observed, but all positive curves under allconditions tested were normal and expressed fluorescence values greaterthan the IgG control, thus confirming that the cells comprise ahomogenous population, which has positive expression of the markers.

EXAMPLE 6 Analysis of Cells by Oligonucleotide Array

Oligonucleotide arrays were used to compare gene expression profiles ofumbilicus- and placenta-derived cells with fibroblasts, humanmesenchymal stem cells, and another cell line derived from human bonemarrow. This analysis provided a characterization of thepostpartum-derived cells and identified unique molecular markers forthese cells.

Materials and Methods

Isolation and Culture of Cells

Postpartum tissue-derived cells. Human umbilical cords and placenta wereobtained from National Disease Research Interchange (NDRI, Philadelphia,Pa.) from normal full term deliveries with patient consent. The tissueswere received and cells were isolated as described in Example 1. Cellswere cultured in Growth Medium on gelatin-coated tissue culture plasticflasks. The cultures were incubated at 37° C. with 5% CO₂.

Fibroblasts. Human dermal fibroblasts were purchased from CambrexIncorporated (Walkersville, Md.; Lot number 9F0844) and ATCC CRL-1501(CCD39SK). Both lines were cultured in DMEM/F12 medium (Invitrogen,Carlsbad, Calif.) with 10% (v/v) fetal bovine serum (Hyclone) andpenicillin/streptomycin (Invitrogen)). The cells were grown on standardtissue-treated plastic.

Human Mesenchymal Stem Cells (hMSC). hMSCs were purchased from CambrexIncorporated (Walkersville, Md.; Lot numbers 2F1655, 2F1656 and 2F1657)and cultured according to the manufacturer's specifications in MSCGMMedia (Cambrex). The cells were grown on standard tissue culturedplastic at 37° C. with 5% CO₂.

Human Ileac Crest Bone Marrow Cells (ICBM). Human ileac crest bonemarrow was received from NDRI with patient consent. The marrow wasprocessed according to the method outlined by Ho, et al. (WO03/025149).The marrow was mixed with lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, and0.1 mM EDTA, pH 7.2) at a ratio of 1 part bone marrow to 20 parts lysisbuffer. The cell suspension was vortexed, incubated for 2 minutes atambient temperature, and centrifuged for 10 minutes at 500×g. Thesupernatant was discarded and the cell pellet was resuspended in MinimalEssential Medium-alpha (Invitrogen) supplemented with 10% (v/v) fetalbovine serum and 4 mM glutamine. The cells were centrifuged again andthe cell pellet was resuspended in fresh medium. The viable mononuclearcells were counted using trypan-blue exclusion (Sigma, St. Louis, Mo.).The mononuclear cells were seeded in tissue-cultured plastic flasks at5×10⁴ cells/cm². The cells were incubated at 37° C. with 5% CO₂ ateither standard atmospheric O₂ or at 5% O₂. Cells were cultured for 5days without a media change. Media and non-adherent cells were removedafter 5 days of culture. The adherent cells were maintained in culture.

Isolation of mRNA and GENECHIP Analysis. Actively growing cultures ofcells were removed from the flasks with a cell scraper in cold phosphatebuffered saline (PBS). The cells were centrifuged for 5 minutes at300×g. The supernatant was removed and the cells were resuspended infresh PBS and centrifuged again. The supernatant was removed and thecell pellet was immediately frozen and stored at −80° C. Cellular mRNAwas extracted and transcribed into cDNA. cDNA was then transcribed intocRNA and biotin-labeled. The biotin-labeled cRNA was hybridized withAffymetrix GENECHP HG-U133A oligonucleotide arrays (Affymetrix, SantaClara, Calif.). The hybridizations and data collection were performedaccording to the manufacturer's specifications. The hybridization anddata collection was performed according to the manufacturer'sspecifications. Data analyses were performed using “SignificanceAnalysis of Microarrays” (SAM) version 1.21 computer software (Tusher,V. G. et al., 2001, Proc. Natl. Acad. Sci. USA 98: 5116-5121). Licensesfor the analysis software are available through the Office of TechnologyLicensing, Stanford University, and more information is available on theWorld Wide Web at Professor Tibshirani's web site in the Dep't ofStatistics, Stanford University (www-stat.stanford.edu/˜tibs/SAM/).

Results

Fourteen different populations of cells were analyzed in this study. Thecells along with passage information, culture substrate, and culturemedia are listed in Table 6-1. TABLE 6-1 Cells analyzed by themicroarray study. The cells lines are listed by their identificationcode along with passage at the time of analysis, cell growth substrate,and growth media. Cell Population Passage Substrate Media Umbilical(022803) 2 Gelatin DMEM, 15% FBS, βME Umbilical (042103) 3 Gelatin DMEM,15% FBS, βME Umbilical (071003) 4 Gelatin DMEM, 15% FBS, βME Placenta(042203) 12 Gelatin DMEM, 15% FBS, βME Placenta (042903) 4 Gelatin DMEM,15% FBS, βME Placenta (071003) 3 Gelatin DMEM, 15% FBS, βME ICBM(070203) (5% O₂) 3 Plastic MEM 10% FBS ICBM (062703) (std O₂) 5 PlasticMEM 10% FBS ICBM (062703)(5% O₂) 5 Plastic MEM 10% FBS hMSC (Lot 2F1655)3 Plastic MSCGM hMSC (Lot 2F1656) 3 Plastic MSCGM hMSC (Lot 2F1657) 3Plastic MSCGM hFibroblast (9F0844) 9 Plastic DMEM-F12, 10% FBShFibroblast (CCD39SK) 4 Plastic DMEM-F12, 10% FBS

The data were evaluated by Principle Component Analysis with SAMsoftware as described above. Analysis revealed 290 genes that wereexpressed in different relative amounts in the cells tested. Thisanalysis provided relative comparisons between the populations.

Table 6-2 shows the Euclidean distances that were calculated for thecomparison of the cell pairs. The Euclidean distances were based on thecomparison of the cells based on the 290 genes that were differentiallyexpressed among the cell types. The Euclidean distance is inverselyproportional to similarity between the expression of the 290 genes.TABLE 6-2 The Euclidean Distances for the Cell Pairs. The Euclideandistance was calculated for the cell types using the 290 genes that wereexpressed differentially between the cell types. Similarity between thecells is inversely proportional to the Euclidean distance. Cell PairEuclidean Distance ICBM-hMSC 24.71 Placenta-umbilical 25.52ICBM-Fibroblast 36.44 ICBM-placenta 37.09 Fibroblast-MSC 39.63ICBM-Umbilical 40.15 Fibroblast-Umbilical 41.59 MSC-Placenta 42.84MSC-Umbilical 46.86 ICBM-placenta 48.41

Tables 6-3, 6-4, and 6-5 show the expression of genes increased inplacenta-derived cells (Table 6-3), increased in umbilical cord-derivedcells (Table 6-4), and reduced in umbilical cord and placenta-derivedcells (Table 6-5). TABLE 6-3 Genes which are specifically increased inexpression in the placenta-derived cells as compared to the other celllines assayed. Genes Increased in Placenta-Derived Cells NCBI AccessionProbe Set ID Gene Name Number 209732_at C-type (calcium dependent,carbohydrate- AF070642 recognition domain) lectin, superfamily member 2(activation-induced) 206067_s_at Wilms tumor 1 NM_024426 207016_s_ataldehyde dehydrogenase 1 family, AB015228 member A2 206367_at ReninNM_000537 210004_at oxidized low density lipoprotein AF035776(lectin-like) receptor 1 214993_at Homo sapiens, clone IMAGE: 4179671,AF070642 mRNA, partial cds 202178_at protein kinase C, zeta NM_002744209780_at hypothetical protein DKFZp564F013 AL136883 204135_atdownregulated in ovarian cancer 1 NM_014890 213542_at Homo sapiens mRNA;cDNA AI246730 DKFZp547K1113 (from clone DKFZp547K1113)

TABLE 6-4 Genes which are specifically increased in expression inumbilical cord - derived cells as compared to the other cell linesassayed. Genes Increased in Umbilicus-Derived Cells NCBI Accession ProbeSet ID Gene Name Number 202859_x_at Interleukin 8 NM_000584 211506_s_atInterleukin 8 AF043337 210222_s_at reticulon 1 BC000314 204470_atchemokine (C-X-C motif) ligand 1 NM_001511 (melanoma growth stimulatingactivity 206336_at chemokine (C-X-C motif) ligand 6 NM_002993(granulocyte chemotactic protein 2) 207850_at Chemokine (C-X-C motif)ligand 3 NM_002090 203485_at reticulon 1 NM_021136 202644_s_at tumornecrosis factor, alpha-induced NM_006290 protein 3

TABLE 6-5 Genes which were decreased in expression in the umbilical cordand placenta cells as compared to the other cell lines assayed. GenesDecreased in Umbilicus- and Placenta-Derived Cells Probe Set NCBIAccession ID Gene name Number 210135_s_at short stature homeobox 2AF022654.1 205824_at heat shock 27 kDa protein 2 NM_001541.1 209687_atchemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)U19495.1 203666_at chemokine (C-X-C motif) ligand 12 (stromalcell-derived factor 1) NM_000609.1 212670_at elastin (supravalvularaortic stenosis, Williams-Beuren syndrome) AA479278 213381_at Homosapiens mRNA; cDNA DKFZp586M2022 (from clone N91149 DKFZp586M2022)206201_s_at mesenchyme homeobox 2 (growth arrest-specific homeobox)NM_005924.1 205817_at Sine oculis homeobox homolog 1 (Drosophila)NM_005982.1 209283_at crystallin, alpha B AF007162.1 212793_atdishevelled associated activator of morphogenesis 2 BF513244 213488_atDKFZP586B2420 protein AL050143.1 209763_at similar to neuralin 1AL049176 205200_at Tetranectin (plasminogen binding protein) NM_003278.1205743_at src homology three (SH3) and cysteine rich domain NM_003149.1200921_s_at B-cell translocation gene 1, anti-proliferative NM_001731.1206932_at cholesterol 25-hydroxylase NM_003956.1 204198_s_atrunt-related transcription factor 3 AA541630 219747_at hypotheticalprotein FLJ23191 NM_024574.1 204773_at Interleukin 11 receptor, alphaNM_004512.1 202465_at Procollagen C-endopeptidase enhancer NM_002593.2203706_s_at Frizzled homolog 7 (Drosophila) NM_003507.1 212736_athypothetical gene BC008967 BE299456 214587_at Collagen, type VIII, alpha1 BE877796 201645_at Tenascin C (hexabrachion) NM_002160.1 210239_atiroquois homeobox protein 5 U90304.1 203903_s_at Hephaestin NM_014799.1205816_at integrin, beta 8 NM_002214.1 203069_at synaptic vesicleglycoprotein 2 NM_014849.1 213909_at Homo sapiens cDNA FLJ12280 fis,clone MAMMA1001744 AU147799 206315_at cytokine receptor-like factor 1NM_004750.1 204401_at potassium intermediate/small conductancecalcium-activated channel, NM_002250.1 subfamily N, member 4 216331_atintegrin, alpha 7 AK022548.1 209663_s_at integrin, alpha 7 AF072132.1213125_at DKFZP586L151 protein AW007573 202133_at transcriptionalco-activator with PDZ-binding motif (TAZ) AA081084 206511_s_at Sineoculis homeobox homolog 2 (Drosophila) NM_016932.1 213435_at KIAA1034protein AB028957.1 206115_at early growth response 3 NM_004430.1213707_s_at distal-less homeobox 5 NM_005221.3 218181_s_at hypotheticalprotein FLJ20373 NM_017792.1 209160_at aldo-keto reductase family 1,member C3 (3-alpha hydroxysteroid AB018580.1 dehydrogenase, type II)213905_x_at Biglycan AA845258 201261_x_at Biglycan BC002416.1 202132_attranscriptional co-activator with PDZ-binding motif (TAZ) AA081084214701_s_at fibronectin 1 AJ276395.1 213791_at Proenkephalin NM_006211.1205422_s_at Integrin, beta-like 1 (with EGF-like repeat domains)NM_004791.1 214927_at Homo sapiens mRNA full length insert cDNA cloneEUROIMAGE AL359052.1 1968422 206070_s_at EphA3 AF213459.1 212805_atKIAA0367 protein AB002365.1 219789_at natriuretic peptide receptorC/guanylate cyclase C (atrionatriuretic AI628360 peptide receptor C)219054_at hypothetical protein FLJ14054 NM_024563.1 213429_at Homosapiens mRNA; cDNA DKFZp564B222 (from clone AW025579 DKFZp564B222)204929_s_at vesicle-associated membrane protein 5 (myobrevin)NM_006634.1 201843_s_at EGF-containing fibulin-like extracellular matrixprotein 1 NM_004105.2 221478_at BCL2/adenovirus E1B 19 kDa interactingprotein 3-like AL132665.1 201792_at AE binding protein 1 NM_001129.2204570_at cytochrome c oxidase subunit VIIa polypeptide 1 (muscle)NM_001864.1 201621_at neuroblastoma, suppression of tumorigenicity 1NM_005380.1 202718_at Insulin-like growth factor binding protein 2, 36kDa NM_000597.1

Tables 6-6, 6-7, and 6-8 show the expression of genes increased in humanfibroblasts (Table 6-6), ICBM cells (Table 6-7), and MSCs (Table 6-8).TABLE 6-6 Genes which were increased in expression in fibroblasts ascompared to the other cell lines assayed. Genes increased in fibroblastsdual specificity phosphatase 2 KIAA0527 protein Homo sapiens cDNA:FLJ23224 fis, clone ADSU02206 dynein, cytoplasmic, intermediatepolypeptide 1 ankyrin 3, node of Ranvier (ankyrin G) inhibin, beta A(activin A, activin AB alpha polypeptide) ectonucleotidepyrophosphatase/phosphodiestrease 4 (putative function) KIAA1053 proteinmicrotubule-associated protein 1A zinc finger protein 41 HSPC019 proteinHomo sapiens cDNA: FLJ23564 fis, clone LNG10773 Homo sapiens mRNA; cDNADKFZp564A072 (from clone DKFZp564A072) LIM protein (similar to ratprotein kinase C-binding enigma) inhibitor of kappa light polypeptidegene enhancer in B-cells, kinase complex-associated protein hypotheticalprotein FLJ22004 Human (clone CTG-A4) mRNA sequence ESTs, Moderatelysimilar to cytokine receptor-like factor 2; cytokine receptor CRL2precursor [Homo sapiens] transforming growth factor, beta 2 hypotheticalprotein MGC29643 antigen identified by monoclonal antibody MRC OX-2putative X-linked retinopathy protein

TABLE 6-7 Genes which were increased in expression in the ICBM-derivedcells as compared to the other cell lines assayed. Genes Increased InICBM Cells cardiac ankyrin repeat protein MHC class I region ORFintegrin, alpha 10 hypothetical protein FLJ22362UDP-N-acetyl-alpha-D-galactosamine:polypeptideN-acetylgalactosaminyltransferase 3 (GalNAc-T3) interferon-inducedprotein 44 SRY (sex determining region Y)-box 9 (campomelic dysplasia,autosomal sex-reversal) keratin associated protein 1-1 hippocalcin-like1 jagged 1 (Alagille syndrome) proteoglycan 1, secretory granule

TABLE 6-8 Genes which were increased in expression in the MSC cells ascompared to the other cell lines assayed. Genes Increased In MSC Cellsinterleukin 26 maltase-glucoamylase (alpha-glucosidase) nuclear receptorsubfamily 4, group A, member 2 v-fos FBJ murine osteosarcoma viraloncogene homolog hypothetical protein DC42 nuclear receptor subfamily 4,group A, member 2 FBJ murine osteosarcoma viral oncogene homolog B WNT1inducible signaling pathway protein 1 MCF.2 cell line derivedtransforming sequence potassium channel, subfamily K, member 15cartilage paired-class homeoprotein 1 Homo sapiens cDNA FLJ12232 fis,clone MAMMA1001206 Homo sapiens cDNA FLJ34668 fis, clone LIVER2000775jun B proto-oncogene B-cell CLL/lymphoma 6 (zinc finger protein 51) zincfinger protein 36, C3H type, homolog (mouse)

Summary. The present study was performed to provide a molecularcharacterization of the postpartum cells derived from umbilical cord andplacenta. This analysis included cells derived from three differentumbilical cords and three different placentas. The study also includedtwo different lines of dermal fibroblasts, three lines of mesenchymalstem cells, and three lines of ileac crest bone marrow cells. The mRNAthat was expressed by these cells was analyzed on a GENECHIPoligonucleotide array that contained oligonucleotide probes for 22,000genes.

The analysis revealed that transcripts for 290 genes were present indifferent amounts in these five different cell types. These genesinclude ten genes that are specifically increased in theplacenta-derived cells and seven genes specifically increased in theumbilical cord-derived cells. Fifty-four genes were found to havespecifically lower expression levels in placenta and umbilical cord.

The expression of selected genes has been confirmed by PCR, as shown inExample 7. Postpartum-derived cells generally, and umbilical derivedcells, in particular, have distinct gene expression profiles, forexample, as compared to other human cells, such as the bonemarrow-derived cells and fibroblasts tested here.

EXAMPLE 7 Cell Markers in Umbilicus-Derived Cells

Gene expression profiles of cells derived from the human umbilical cordwere compared with those of cells derived from other sources using anAffymetrix GENECHIP. Six “signature” genes were identified: oxidized LDLreceptor 1, interleukin-8 (IL-8), renin, reticulon, chemokine receptorligand 3 (CXC ligand 3), and granulocyte chemotactic protein 2 (GCP-2).These “signature” genes were expressed at relatively high levels inumbilicus-derived cells.

The procedures described in this example were conducted to verify themicroarray data and compare data for gene and protein expression, aswell as to establish a series of reliable assays for detection of uniqueidentifiers for umbilicus-derived cells.

Methods & Materials

Cells. Umbilicus-derived cells (four isolates), and Normal Human DermalFibroblasts (NHDF; neonatal and adult) were grown in Growth Medium ingelatin-coated T75 flasks. Mesenchymal Stem Cells (MSCs) were grown inMesenchymal Stem Cell Growth Medium Bullet kit (MSCGM; Cambrex,Walkerville, Md.).

For IL-8 experiments, cells were thawed from liquid nitrogen and platedin gelatin-coated flasks at 5,000 cells/cm², grown for 48 hours inGrowth Medium and then grown further for 8 hours in 10 milliliters ofserum starvation medium [DMEM-low glucose (Gibco, Carlsbad, Calif.),penicillin (50 Units/milliliter), streptomycin (50micrograms/milliliter)(Gibco) and 0.1% (w/v) Bovine Serum Albumin (BSA;Sigma, St. Louis, Mo.)]. RNA was then extracted and the supernatantswere centrifuged at 150×g for 5 minutes to remove cellular debris.Supernatants were frozen at −80° C. until ELISA analysis.

Cell culture for ELISA assay. Cells derived from human umbilical cord,as well as human fibroblasts derived from human neonatal foreskin, werecultured in Growth Medium in gelatin-coated T75 flasks. Cells werefrozen at passage 11 in liquid nitrogen. Cells were thawed andtransferred to 15 milliliter centrifuge tubes. After centrifugation at150×g for 5 minutes, the supernatant was discarded. Cells wereresuspended in 4 milliliters culture medium and counted. Cells weregrown in a 75 cm² flask containing 15 milliliters of Growth Medium at375,000 cell/flask for 24 hours. The medium was changed to a serumstarvation medium for 8 hours. Serum starvation medium was collected atthe end of incubation, centrifuged at 14,000×g for 5 minutes (and storedat −20° C.).

To estimate the number of cells in each flask, 2 milliliters oftrypsin/EDTA (Gibco, Carlsbad, Calif.) were added each flask. Aftercells detached from the flask, trypsin activity was neutralized with 8milliliters of Growth Medium. Cells were transferred to a 15 millilitercentrifuge tube and centrifuged at 150×g for 5 minutes. Supernatant wasremoved and 1 milliliter Growth Medium was added to each tube toresuspend the cells. Cell number was determined with a hemocytometer.

ELISA assay. The amount of IL-8 secreted by the cells into serumstarvation medium was analyzed using ELISA assays (R&D Systems,Minneapolis, Minn.). All assays were conducted according to theinstructions provided by the manufacturer.

Total RNA isolation. RNA was extracted from confluent umbilicus-derivedcells and fibroblasts, or for IL-8 expression, from cells treated asdescribed above. Cells were lysed with 350 microliters buffer RLTcontaining beta-mercaptoethanol (Sigma, St. Louis, Mo.) according to themanufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia, Calif.).RNA was extracted according to the manufacturer's instructions (RNeasyMini Kit; Qiagen, Valencia, Calif.) and subjected to DNase treatment(2.7 Units/sample) (Sigma St. Louis, Mo.). RNA was eluted with 50microliters DEPC-treated water and stored at −80° C. RNA was alsoextracted from human umbilical cord. Tissue (30 milligrams) wassuspended in 700 microliters of buffer RLT containingbeta-mercaptoethanol. Samples were mechanically homogenized and the RNAextraction proceeded according to manufacturer's specification. RNA wasextracted with 50 microliters of DEPC-treated water and stored at −80°C.

Reverse transcription. RNA was reverse-transcribed using random hexamerswith the TaqMan reverse transcription reagents (Applied Biosystems,Foster City, Calif.) at 25° C. for 10 minutes, 37° C. for 60 minutes,and 95° C. for 10 minutes. Samples were stored at −20° C.

Genes identified by cDNA microarray as uniquely regulated in postpartumcells (signature genes—including oxidized LDL receptor, interleukin-8,renin, and reticulon), were further investigated using real-time andconventional PCR.

Real-time PCR. PCR was performed on cDNA samples using gene expressionproducts sold under the tradename ASSAYS-ON-DEMAND (Applied Biosystems)gene expression products. Oxidized LDL receptor (Hs00234028); renin(Hs00166915); reticulon (Hs00382515); CXC ligand 3 (Hs00171061); GCP-2(Hs00605742); IL-8 (Hs00174103); and GAPDH were mixed with cDNA andTaqMan Universal PCR master mix according to the manufacturer'sinstructions (Applied Biosystems) using a 7000 sequence detection systemwith ABI Prism 7000 SDS software (Applied Biosystems). Thermal cycleconditions were initially 50° C. for 2 minutes and 95° C. for 10minutes, followed by 40 cycles of 95° C. for 15 seconds and 60° C. for 1minute. PCR data were analyzed according to manufacturer'sspecifications (User Bulletin #2 from Applied Biosystems for ABI Prism7700 Sequence Detection System).

Conventional PCR. Conventional PCR was performed using an ABI PRISM 7700(Perkin Elmer Applied Biosystems, Boston, Mass.) to confirm the resultsfrom real-time PCR. PCR was performed using 2 microliters of cDNAsolution (1×Taq polymerase (tradename AMPLITAQ GOLD) universal mix PCRreaction buffer (Applied Biosystems) and initial denaturation at 94° C.for 5 minutes. Amplification was optimized for each primer set. ForIL-8, CXC ligand 3, and reticulon (94° C. for 15 seconds, 55° C. for 15seconds and 72° C. for 30 seconds for 30 cycles); for renin (94° C. for15 seconds, 53° C. for 15 seconds and 72° C. for 30 seconds for 38cycles); for oxidized LDL receptor and GAPDH (94° C. for 15 seconds, 55°C. for 15 seconds and 72° C. for 30 seconds for 33 cycles). Primers usedfor amplification are listed in Table 7-1. Primer concentration in thefinal PCR reaction was 1 micromolar except for GAPDH which was 0.5micromolar. GAPDH primers were the same as for real-time PCR, exceptthat the manufacturer's TaqMan probe was not added to the final PCRreaction. Samples were separated on 2% (w/v) agarose gel and stainedwith ethidium bromide (Sigma, St. Louis, Mo.). Images were captured on667 film (Universal Twinpack, VWR International, South Plainfield, N.J.)using a fixed focal-length POLAROID camera (VWR International, SouthPlainfield, N.J.). TABLE 7-1 Primers used Primer name Primers OxidizedLDL S: 5′-GAGAAATCCAAAGAGCAAATGG-3′ receptor (SEQ ID NO:1) A:5′-AGAATGGAAAACTGGAATAGG-3′ (SEQ ID NO:2) Renin S:5′-TCTTCGATGCTTCGGATTCC-3′ (SEQ ID NO:3) A: 5′-GAATTCTCGGAATCTCTGTTG-3′(SEQ ID NO:4) Reticulon S: 5′-TTACAAGCAGTGCAGAAAACC-3′ (SEQ ID NO:5) A:5′-AGTAAACATTGAAACCACAGCC-3′ (SEQ ID NO:6) Interleukin-8 S:5′-TCTGCAGCTCTGTGTGAAGG-3′ (SEQ ID NO:7) A: 5′-CTTCAAAAACTTCTCCACAACC-3′(SEQ ID NO:8) Chemokine (CXC) S: 5′-CCCACGCCACGCTCTCC-3′ ligand 3 (SEQID NO:9) A: 5′-TCCTGTCAGTTGGTGCTCC-3′ (SEQ ID NO:10)

Immunofluorescence. Cells were fixed with cold 4% (w/v) paraformaldehyde(Sigma-Aldrich, St. Louis, Mo.) for 10 minutes at room temperature.Umbilicus-derived cells at passage 0 (P0) (one isolate, directly afterisolation) and passage 11 (P11) (two isolates of umbilicus-derivedcells), and fibroblasts (P11) were used. Immunocytochemistry wasperformed using antibodies directed against the following epitopes:vimentin (1:500, Sigma, St. Louis, Mo.), desmin ((Sigma) 1:150; raisedagainst rabbit; or (Chemicon, Temecula, Calif.) 1:300, raised againstmouse), alpha-smooth muscle actin (SMA; 1:400; Sigma), cytokeratin 18(CK18; 1:400; Sigma), von Willebrand Factor (vWF; 1:200; Sigma), andCD34 (human CD34 Class III; 1:100; DAKOCytomation, Carpinteria, Calif.).In addition, the following markers were tested on passage 11 postpartumcells: anti-human GROalpha—PE (1:100; Becton Dickinson, Franklin Lakes,N.J.), anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, Calif.),anti-human oxidized LDL receptor 1 (ox-LDL R1; 1:100; Santa CruzBiotech), and anti-human NOGO-A (1:100; Santa Cruz, Biotech).

Cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma, St. Louis, Mo.) for 30 minutes to access intracellular antigens.Where the epitope of interest was located on the cell surface (CD34,ox-LDL R1), Triton X-100 was omitted in all steps of the procedure inorder to prevent epitope loss. Furthermore, in instances where theprimary antibody was raised against goat (GCP-2, ox-LDL RI, NOGO-A), 3%(v/v) donkey serum was used in place of goat serum throughout. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. The primaryantibody solutions were removed and the cultures were washed with PBSprior to application of secondary antibody solutions (1 hour at roomtemperature) containing block along with goat anti-mouse IgG-Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and/or goat anti-rabbitIgG-Alexa 488 (1:250; Molecular Probes) or donkey anti-goat IgG-FITC(1:150, Santa Cruz Biotech). Cultures were then washed and 10 micromolarDAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using anappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution (no 1° control). Representative imageswere captured using a digital color videocamera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

FACS analysis. Adherent cells in flasks were washed in phosphatebuffered saline (PBS) (Gibco, Carlsbad, Calif.) and detached withTrypsin/EDTA (Gibco, Carlsbad, Calif.). Cells were harvested,centrifuged, and resuspended 3% (v/v) FBS in PBS at a concentration of1×10⁷ cells/milliliter. One hundred microliter aliquots were deliveredto conical tubes. Cells stained for intracellular antigens werepermeabilized with Perm/Wash buffer (BD Pharmingen, San Diego, Calif.).Antibody was added to aliquots as per manufactures specifications andthe cells were incubated for in the dark for 30 minutes at 4° C. Afterincubation, cells were washed with PBS and centrifuged to remove excessantibody. Cells requiring a secondary antibody were resuspended in 100microliters of 3% FBS. Secondary antibody was added as per manufacturesspecification and the cells were incubated in the dark for 30 minutes at4° C. After incubation, cells were washed with PBS and centrifuged toremove excess secondary antibody. Washed cells were resuspended in 0.5milliliter PBS and analyzed by flow cytometry. The following antibodieswere used: oxidized LDL receptor 1 (sc-5813; Santa Cruz, Biotech),GROalpha (555042; BD Pharmingen, Bedford, Mass.), Mouse IgG1 kappa,(P-4685 and M-5284; Sigma), Donkey against Goat IgG (sc-3743; SantaCruz, Biotech.). Flow cytometry analysis was performed with FACSCalibur(Becton Dickinson San Jose, Calif.).

Results

Results of real-time PCR for selected “signature” genes performed oncDNA from cells derived from human umbilical cord, adult and neonatalfibroblasts, and Mesenchymal Stem Cells (MSCs) indicate that bothreticulon and oxidized LDL receptor expression were higher inumbilicus-derived cells as compared to other cells. The data obtainedfrom real-time PCR were analyzed by the AACT method and expressed on alogarithmic scale. No significant differences in the expression levelsof CXC ligand 3 and GCP-2 were found between postpartum cells andcontrols. The results of real-time PCR were confirmed by conventionalPCR. Sequencing of PCR products further validated these observations. Nosignificant difference in the expression level of CXC ligand 3 was foundbetween postpartum cells and controls using conventional PCR CXC ligand3 primers listed in Table 7-1.

The expression of the cytokine, IL-8 in postpartum cells was elevated inboth Growth Medium-cultured and serum-starved postpartum-derived cells.All real-time PCR data were validated with conventional PCR and bysequencing PCR products.

After growth in serum-free media, the conditioned media were examinedfor the presence of IL-8. The greatest amounts of IL-8 were detected inmedia in which umbilical cells had been grown (Table 7-2). No IL-8 wasdetected in medium in which human dermal fibroblasts had been grown.TABLE 7-2 IL-8 protein expression measured by ELISA Cell type IL-8secretion hFibro ND UMBC Isolate 1 2058.42 ± 144.67 UMBC Isolate 22368.86 ± 22.73 Results of the ELISA assay for interleukin-8 (IL-8) performed on spentmedia in which had been grown umbilicus-derived cells and human skinfibroblasts. Values are presented here are pg/million cells, n = 2, sem.ND: Not Detected

Cells derived from the human umbilical cord at passage 0 were probed forthe expression of selected proteins by immunocytochemical analysis.Immediately after isolation (passage 0), cells were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Umbilicus-derived cells were positive foralpha-smooth muscle actin and vimentin, with the staining patternconsistent through passage 11.

The expression of GROalpha, GCP-2, oxidized LDL receptor 1 and reticulonin umbilicus-derived cells at passage 11 was investigated byimmunocytochemistry.

Summary. Concordance between gene expression levels measured bymicroarray and PCR (both real-time and conventional) has beenestablished for four genes: oxidized LDL receptor 1, renin, reticulon,and IL-8. The expression of these genes was regulated at the mRNA levelin postpartum cells. IL-8 was also regulated at the protein level.Differences in expression of GCP-2 and CXC ligand 3 were not confirmedat the mRNA level.

Cells derived from the human umbilical cord at passage 0 were probed forthe expression of alpha-smooth muscle actin and vimentin, and werepositive for both. The staining pattern was preserved through passage11, suggesting that expression of vimentin and alpha-smooth muscle actinare preserved in cells with passaging, at least in the Growth Mediumused.

EXAMPLE 8 Immunohistochemical Characterization of Umbilical CordCellPhenotypes

The phenotypes of cells found within human umbilical cord tissue wasanalyzed by immunohistochemistry.

Materials & Methods

Tissue Preparation. Human umbilical cord tissue was harvested andimmersion fixed in 4% (w/v) paraformaldehyde overnight at 4° C.Immunohistochemistry was performed using antibodies directed against thefollowing epitopes (see Table 8-1): vimentin (1:500; Sigma, St. Louis,Mo.), desmin (1:150, raised against rabbit; Sigma; or 1:300, raisedagainst mouse; Chemicon, Temecula, Calif.), alpha-smooth muscle actin(SMA; 1:400; Sigma), cytokeratin 18 (CK18; 1:400; Sigma), von WillebrandFactor (vWF; 1:200; Sigma), and CD34 (human CD34 Class III; 1:100;DAKOCytomation, Carpinteria, Calif.). In addition, the following markerswere tested: anti-human GROalpha-PE (1:100; Becton Dickinson, FranklinLakes, N.J.), anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz,Calif.), anti-human oxidized LDL receptor 1 (ox-LDL R1; 1:100; SantaCruz Biotech), and anti-human NOGO-A (1:100; Santa Cruz Biotech). Fixedspecimens were trimmed with a scalpel and placed within OCT embeddingcompound (Tissue-Tek OCT; Sakura, Torrance, Calif.) on a dry ice bathcontaining ethanol. Frozen blocks were then sectioned (10 microns thick)using a standard cryostat (Leica Microsystems) and mounted onto glassslides for staining.

Immunohistochemistry. Immunohistochemistry was performed similar toprevious studies (e.g., Messina, et al. (2003) Exper. Neurol. 184:816-829). Tissue sections were washed with phosphate-buffered saline(PBS) and exposed to a protein blocking solution containing PBS, 4%(v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton(Triton X-100; Sigma) for 1 hour to access intracellular antigens. Ininstances where the epitope of interest would be located on the cellsurface (CD34, ox-LDL R1), triton was omitted in all steps of theprocedure in order to prevent epitope loss. Furthermore, in instanceswhere the primary antibody was raised against goat (GCP-2, ox-LDL R1,NOGO-A), 3% (v/v) donkey serum was used in place of goat serumthroughout the procedure. Primary antibodies, diluted in blockingsolution, were then applied to the sections for a period of 4 hours atroom temperature. Primary antibody solutions were removed, and cultureswashed with PBS prior to application of secondary antibody solutions (1hour at room temperature) containing block along with goat anti-mouseIgG-Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and/or goatanti-rabbit IgG-Alexa 488 (1:250; Molecular Probes) or donkey anti-goatIgG-FITC (1:150; Santa Cruz Biotech). Cultures were washed, and 10micromolar DAPI (Molecular Probes) was applied for 10 minutes tovisualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epifluorescentmicroscope (Olympus, Melville, N.Y.). Positive staining was representedby fluorescence signal above control staining. Representative imageswere captured using a digital color videocamera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.). TABLE 8-1 Summary of Primary Antibodies Used AntibodyConcentration Vendor Vimentin 1:500 Sigma, St. Louis, MO Desmin (rb)1:150 Sigma Desmin (m) 1:300 Chemicon, Temecula, CA alpha-smooth muscleactin 1:400 Sigma (SMA) Cytokeratin 18 (CK18) 1:400 Sigma von Willebrandfactor 1:200 Sigma (vWF) CD34 III 1:100 DakoCytomation, Carpinteria, CAGROalpha-PE 1:100 BD, Franklin Lakes, NJ GCP-2 1:100 Santa Cruz BiotechOx-LDL R1 1:100 Santa Cruz Biotech NOGO-A 1:100 Santa Cruz BiotechResults

Umbilical Cord Characterization. Vimentin, desmin, SMA, CK18, vWF, andCD34 markers were expressed in a subset of the cells found withinumbilical cord. In particular, vWF and CD34 expression were restrictedto blood vessels contained within the cord. CD34+ cells were on theinnermost layer (lumen side). Vimentin expression was found throughoutthe matrix and blood vessels of the cord. SMA was limited to the matrixand outer walls of the artery and vein, but not contained with thevessels themselves. CK18 and desmin were observed within the vesselsonly, desmin being restricted to the middle and outer layers.

Summary. Vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18,von Willebrand Factor, and CD34 are produced in cells within humanumbilical cord.

EXAMPLE 9 Secretion of Trophic Factors by Umbilicus-Derived Cells

The secretion of selected trophic factors from umbilicus-derived PPDCswas measured. Factors were selected that have angiogenic activity (i.e.,hepatocyte growth factor (HGF) (Rosen et al. (1997) Ciba Found. Symp.212:215-26), monocyte chemotactic protein 1 (MCP-1) (Salcedo et al.(2000) Blood 96;34-40), interleukin-8 (IL-8) (Li et al. (2003) J.Immunol. 170:3369-76), keratinocyte growth factor (KGF), basicfibroblast growth factor (bFGF), vascular endothelial growth factor(VEGF) (Hughes et al. (2004) Ann. Thorac. Surg. 77:812-8), tissueinhibitor of matrix metalloproteinase 1 (TIMP1), angiopoietin 2 (ANG2),platelet derived growth factor (PDGFbb), thrombopoietin (TPO),heparin-binding epidermal growth factor (HB-EGF), stromal-derived factor1alpha (SDF-1alpha)), neurotrophic/neuroprotective activity(brain-derived neurotrophic factor (BDNF) (Cheng et al. (2003) Dev.Biol. 258;319-33), interleukin-6 (IL-6), granulocyte chemotacticprotein-2 (GCP-2), transforming growth factor beta2 (TGFbeta2)), orchemokine activity (macrophage inflammatory protein 1alpha (MIPlalpha),macrophage inflammatory protein 1 beta (MIP1beta), monocytechemoattractant-1 (MCP-1), Rantes (regulated on activation, normal Tcell expressed and secreted), I309, thymus and activation-regulatedchemokine (TARC), Eotaxin, macrophage-derived chemokine (MDC), IL-8).

Methods & Materials

Cell culture. PPDCs derived from umbilical cord, as well as humanfibroblasts derived from human neonatal foreskin, were cultured inGrowth Medium on gelatin-coated T75 flasks. Cells were cryopreserved atpassage 11 and stored in liquid nitrogen. After thawing, Growth Mediumwas added to the cells, followed by transfer to a 15 millilitercentrifuge tube and centrifugation of the cells at 150×g for 5 minutes.The cell pellet was resuspended in 4 milliliters Growth Medium, andcells were counted. Cells were seeded at 5,000 cells/cm² in T75 flaskseach containing 15 milliliters of Growth Medium, and cultured for 24hours. The medium was changed to a serum-free medium (DMEM-low glucose(Gibco), 0.1% (w/v) bovine serum albumin (Sigma), penicillin (50Units/milliliter) and streptomycin (50 micrograms/milliliter, Gibco))for 8 hours. Conditioned serum-free medium was collected at the end ofincubation by centrifugation at 14,000×g for 5 minutes and stored at−20° C.

To estimate the number of cells in each flask, cells were washed withphosphate-buffered saline (PBS) and detached using 2 milliliterstrypsin/EDTA (Gibco). Trypsin activity was inhibited by addition of 8milliliters Growth Medium. Cells were centrifuged at 150×g for 5minutes. The supernatant was removed, and cells were resuspended in 1milliliter Growth Medium. Cell number was estimated with ahemocytometer.

ELISA assay. Cells were grown at 37° C. in 5% carbon dioxide andatmospheric oxygen. The amount of MCP-1, IL-6, VEGF, SDF-1alpha, GCP-2,IL-8, and TGF-beta2 produced by each cell sample was determined by ELISA(R&D Systems, Minneapolis, Minn.). All assays were performed accordingto the manufacturer's instructions. Values presented are picograms permilliliter per million cells (n=2, sem).

SearchLight Multiplexed ELISA assay. Chemokines (MIP1alpha, MIP1beta,MCP-1, Rantes, 1309, TARC, Eotaxin, MDC, IL8), BDNF, and angiogenicfactors (HGF, KGF, bFGF, VEGF, TIMP1, ANG2, PDGFbb, TPO, HB-EGF weremeasured using SearchLight Proteome Arrays (Pierce Biotechnology Inc.).The Proteome Arrays are multiplexed sandwich ELISAs for the quantitativemeasurement of two to sixteen proteins per well. The arrays are producedby spotting a 2×2, 3×3, or 4×4 pattern of four to sixteen differentcapture antibodies into each well of a 96-well plate. Following asandwich ELISA procedure, the entire plate is imaged to capture thechemiluminescent signal generated at each spot within each well of theplate. The signal generated at each spot is proportional to the amountof target protein in the original standard or sample.

Results

ELISA assay. MCP-1 and IL-6 were secreted by umbilicus-derived PPDCs anddermal fibroblasts (Table 9-1). SDF-1alpha and GCP-2 were secreted byfibroblasts. GCP-2 and IL-8 were secreted by umbilicus-derived PPDCs.TGF-beta2 was not detected from either cell type by ELISA. TABLE 9-1ELISA Results: Detection of Trophic Factors MCP-1 IL-6 VEGF SDF-1α GCP-2IL-8 TGF-beta2 Fibroblast  17 ± 1 61 ± 3 29 ± 2 19 ± 1 21 ± 1 ND NDUmbilical (022803) 1150 ± 74 4234 ± 289 ND ND 160 ± 11 2058 ± 145 NDUmbilical (071003) 2794 ± 84 1356 ± 43  ND ND 2184 ± 98  2369 ± 23  NDKey:ND: Not Detected.,=/− sem

SearchLight Multiplexed ELISA assay. TIMP1, TPO, KGF, HGF, FGF, HBEGF,BDNF, MIP1beta, MCP1, RANTES, 1309, TARC, MDC, and IL-8 were secretedfrom umbilicus-derived PPDCs (Tables 9-2 and 9-3). No Ang2, VEGF, orPDGFbb were detected. TABLE 9-2 SearchLight Multiplexed ELISA assayresults TIMP1 ANG2 PDGFbb TPO KGF HGF FGF VEGF HBEGF BDNF hFB 19306.3 NDND 230.5 5.0 ND ND 27.9 1.3 ND U1 57718.4 ND ND 1240.0 5.8 559.3 148.7ND 9.3 165.7 U3 21850.0 ND ND 1134.5 9.0 195.6  30.8 ND 5.4 388.6Key:hFB (human fibroblasts),U1 (umbilicus-derived PPDC (022803)),U3 (umbilicus-derived PPDC (071003)).ND: Not Detected.

TABLE 9-3 SearchLight Multiplexed ELISA assay results MIP1a MIP1b MCP1RANTES I309 TARC Eotaxin MDC IL8 hFB ND ND 39.6 ND ND 0.1 ND ND 204.9 U1ND 8.0 1694.2 ND 22.4 37.6 ND 18.9 51930.1 U3 ND 5.2 2018.7 41.5 11.621.4 ND  4.8 10515.9Key:hFB (human fibroblasts),U1 (umbilicus-derived PPDC (022803)),U3 (umbilicus-derived PPDC (071003)).ND: Not Detected.

Summary. Umbilicus-derived cells secreted a number of trophic factors.Some of these trophic factors, such as HGF, bFGF, MCP-1 and IL-8, playimportant roles in angiogenesis. Other trophic factors, such as BDNF andIL-6, have important roles in neural regeneration or protection.

EXAMPLE 10 In Vitro Immunology

Postpartum cell lines were evaluated in vitro for their immunologicalcharacteristics in an effort to predict the immunological response, ifany, these cells would elicit upon in vivo transplantation. Postpartumcell lines were assayed by flow cytometry for the expression of HLA-DR,HLA-DP, HLA-DQ, CD80, CD86, and B7-H2. These proteins are expressed byantigen-presenting cells (APC) and are required for the directstimulation of naïve CD4⁺ T cells (Abbas & Lichtman, CELLULAR ANDMOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171).The cell lines were also analyzed by flow cytometry for the expressionof HLA-G (Abbas & Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th Ed.(2003) Saunders, Philadelphia, p. 171), CD178 (Coumans, et. al., (1999)Journal of Immunological Methods 224, 185-196), and PD-L2 (Abbas &Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders,Philadelphia, p. 171; Brown, et. al. (2003) The Journal of Immunology170, 1257-1266). The expression of these proteins by cells residing inplacental tissues is thought to mediate the immuno-privileged status ofplacental tissues in utero. To predict the extent to which postpartumumbilicus-derived cell lines elicit an immune response in vivo, the celllines were tested in a one-way mixed lymphocyte reaction (MLR).

Materials and Methods

Cell culture. Cells were cultured in Growth Media in T75 flasks(Corning, Corning, N.Y.) coated with 2% gelatin (Sigma, St. Louis, Mo.)until confluent.

Antibody Staining. Cells were washed in phosphate buffered saline (PBS)(Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco,Carlsbad, Mo.). Cells were harvested, centrifuged, and resuspended in 3%(v/v) FBS in PBS at a cell concentration of 1×10⁷ per milliliter.Antibody (Table 10-1) was added to one hundred microliters of cellsuspension as per manufacturer's specifications and incubated in thedark for 30 minutes at 4° C. After incubation, cells were washed withPBS and centrifuged to remove unbound antibody. Cells were re-suspendedin five hundred microliters of PBS and analyzed by flow cytometry usinga FACSCalibur instrument (Becton Dickinson, San Jose, Calif.). TABLE10-1 Antibodies Catalog Antibody Manufacture Number HLA-DR, DP, DQ BDPharmingen (San Diego, CA) 555558 CD80 BD Pharmingen 557227 CD86 BDPharmingen 555665 B7-H2 BD Pharmingen 552502 HLA-G Abcam(Cambridgeshire, UK) ab 7904-100 CD178 Santa Cruz (San Cruz, CA)sc-19681 PD-L2 BD Pharmingen 557846 Mouse IgG2alpha Sigma (St. Louis,MO) F-6522 Mouse IgG1kappa Sigma P-4685

Mixed Lymphocyte Reaction. Cryopreserved vials of passage 10umbilicus-derived PPDCs labeled as cell line “A” were sent on dry ice toCTBR (Senneville, Quebec) to conduct a mixed lymphocyte reaction usingCTBR SOP no. CAC-031. Peripheral blood mononuclear cells (PBMCs) werecollected from multiple male and female volunteer donors. Six humanvolunteer blood donors were screened to identify a single allogeneicdonor that exhibited a robust proliferation response in a mixedlymphocyte reaction with the other five blood donors. This donor wasselected as the allogeneic positive control donor. The remaining fiveblood donors were selected as recipients. Stimulator (donor) allogeneicPBMC, autologous PBMC, and postpartum cell lines were treated withmitomycin C. Autologous and mitomycin C-treated stimulator cells wereadded to responder (recipient) PBMCs and cultured for 4 days. Afterincubation, [³H]thymidine was added to each sample and cultured for 18hours. Following harvest of the cells, radiolabeled DNA was extracted,and [³H]-thymidine incorporation was measured using a scintillationcounter. Reactions were performed in triplicate using two-cell cultureplates with three receivers per plate

The stimulation index for the allogeneic donor (SIAD) was calculated asthe mean proliferation of the receiver plus mitomycin C-treatedallogeneic donor divided by the baseline proliferation of the receiver.The stimulation index of the postpartum cells was calculated as the meanproliferation of the receiver plus mitomycin C-treated postpartum cellline divided by the baseline proliferation of the receiver.

Results

Mixed Lymphocyte Reaction-Umbilicus-Derived Cells. Results are shownbelow in Tables 10-2 and -3. The average stimulation index ranged from6.5 (plate 1) to 9 (plate 2) and the allogeneic donor positive controlsranged from 42.75 (plate 1) to 70 (plate 2) (Table 10-3). TABLE 10-2Mixed Lymphocyte Reaction Data - Cell Line A (Umbilicus) DPM forProliferation Assay Analytical Culture Replicates number System 1 2 3Mean SD CV Plate ID: Plate 1 IM04-2478 Proliferation baseline ofreceiver 1074 406 391 623.7 390.07 62.5 Control of autostimulation(Mitomycin C treated autologous cells) 672 510 1402 861.3 475.19 55.2MLR allogenic donor IM04-2477 (Mitomycin C treated) 43777 48391 3823143466.3 5087.12 11.7 MLR with cell line (Mitomycin C treated cell typeA) 2914 5622 6109 4881.7 1721.36 35.3 SI (donor) 70 SI (cell line) 8IM04-2479 Proliferation baseline of receiver 530 508 527 521.7 11.93 2.3Control of autostimulation (Mitomycin C treated autologous cells) 701567 1111 793.0 283.43 35.7 MLR allogenic donor IM04-2477 (Mitomycin Ctreated) 25593 24732 22707 24344.0 1481.61 6.1 MLR with cell line(Mitomycin C treated cell type A) 5086 3932 1497 3505.0 1832.21 52.3 SI(donor) 47 SI (cell line) 7 IM04-2480 Proliferation baseline of receiver1192 854 1330 1125.3 244.90 21.8 Control of autostimulation (Mitomycin Ctreated autologous cells) 2963 993 2197 2051.0 993.08 48.4 MLR allogenicdonor IM04-2477 (Mitomycin C treated) 25416 29721 23757 26298.0 3078.2711.7 MLR with cell line (Mitomycin C treated cell type A) 2596 5076 34263699.3 1262.39 34.1 SI (donor) 23 SI (cell line) 3 IM04-2481Proliferation baseline of receiver 695 451 555 567.0 122.44 21.6 Controlof autostimulation (Mitomycin C treated autologous cells) 738 1252 464818.0 400.04 48.9 MLR allogenic donor IM04-2477 (Mitomycin C treated)13177 24885 15444 17835.3 6209.52 34.8 MLR with cell line (Mitomycin Ctreated cell type A) 4495 3671 4674 4280.0 534.95 12.5 SI (donor) 31 SI(cell line) 8 Plate ID: Plate 2 IM04-2482 Proliferation baseline ofreceiver 432 533 274 413.0 130.54 31.6 Control of autostimulation(Mitomycin C treated autologous cells) 1459 633 598 896.7 487.31 54.3MLR allogenic donor IM04-2477 (Mitomycin C treated) 24286 30823 3134628818.3 3933.82 13.7 MLR with cell line (Mitomycin C treated cell typeA) 2762 1502 6723 3662.3 2724.46 74.4 SI (donor) 70 SI (cell line) 9IM04-2477 Proliferation baseline of receiver 312 419 349 360.0 54.3415.1 (allogenic Control of autostimulation (Mitomycin treated autologouscells) 567 604 374 515.0 123.50 24.0 donor) Cell line Proliferationbaseline of receiver 5101 3735 2973 3936.3 1078.19 27.4 type A Controlof autostimulation (Mitomycin treated autologous cells) 1924 4570 21532882.3 1466.04 50.9

TABLE 10-3 Average stimulation index of umbilical cells and anallogeneic donor in a mixed lymphocyte reaction with five individualallogeneic receivers. Average Stimulation Index Recipient UmbilicusPlate 1 (receivers 1-4) 42.75 6.5 Plate 2 (receiver 5) 70 9

Antigen Presenting Cell Markers Produced by Umbilicus-Derived Cells. Thehistograms of the flow cytometry analysis show umbilical cells werenegative for production of HLA-DR, DP, DQ, CD80, CD86, and B7-H2,because the fluorescence values were comparable to the IgG control. Thisindicates that umbilical cell lines lack the cell surface moleculesrequired to directly stimulate CD4⁺ T cells.

Immuno-modulating Markers in Umbilicus-Derived Cells. The umbilicalcells analyzed by flow cytometry were positive for expression of PD-L2,as reflected in the increase in fluorescence relative to the IgGcontrol. The cells were negative for expression of CD178 and HLA-G, asnoted by fluorescence values consistent with the IgG control.

Summary. In the mixed lymphocyte reactions conducted with umbilical celllines the average stimulation index ranged from 6.5 to 9, while that ofthe allogeneic positive controls ranged from 42.75 to 70. Umbilical celllines did not express detectable amounts of the stimulating proteinsHLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2, as measured by flowcytometry. Umbilical cell lines also did not express theimmuno-modulating proteins HLA-G and CD178, but expression of PD-L2 wasdetected by flow cytometry. Allogeneic donor PBMCs containantigen-presenting cells expressing HLA-DR, DQ, CD8, CD86, and B7-H2,thereby allowing for the stimulation of allogeneic lymphocytes. Theabsence on umbilicus-derived cells of antigen-presenting cell surfacemolecules required for the direct stimulation of naïve CD4⁺ T cells, aswell as the presence of PD-L2, an immunomodulating protein, couldaccount for the low stimulation index exhibited by these cells in a MLRas compared to allogeneic controls.

EXAMPLE 11 Plasma Clotting Assay

Cells useful for therapy may be injected systemically for certainapplications where the cells are able to target the site of action. Itis important that injected cells not cause thrombosis, as it may befatal. Tissue factor, a membrane-bound procoagulant glycoprotein, is theinitiator of the extrinsic clotting cascade, which is the predominantcoagulation pathway in vivo. Tissue factor also plays an important rolein embryonic vessel formation, for example, in the formation of theprimitive vascular wall (Brodsky et al. (2002) Exp. Nephrol.10:299-306). To determine the potential for PPDCs to initiate clotting,umbilicus-derived PPDCs were evaluated for tissue factor expression andfor their ability to initiate plasma clotting.

Methods & Materials

Human Tissue factor. Human tissue factor (SIMPLASTIN, Organon TeknikaCorporation, Durham, N.C.), was reconstituted with 20 millilitersdistilled water. The stock solution was serially diluted (1:2) in eighttubes. Normal human plasma (George King Bio-Medical, Overland Park,Kans.) was thawed at 37° C. in a water bath and then stored in icebefore use. 100 microliters phosphate buffered saline (PBS), 10microliters diluted SIMPLASTIN, 30 microliters 0.1 Molar calciumchloride, and 100 microliters of normal human plasma were added to eachwell of a 96-well plate. A negative control well did not receive anySIMPLASTIN. The plate was immediately placed in a temperature-controlledmicroplate reader and absorbance measured at 405 nanometer at 40 secondintervals for 30 minutes.

J-82 and umbilicus-derived cells. J-82 cells (ATCC, MD) were grown inIscove's modified Dulbecco's medium (IMDM; Gibco, Carlsbad, Calif.)containing 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan Utah), 1millimolar sodium pyruvate (Sigma Chemical, St. Louis, Mo.), 2millimolar L-Glutamine (Mediatech Herndon, Va.), 1× non-essential aminoacids (Mediatech Herndon, Va.). At about 70% confluence, cells weretransferred at 100,000, 50,000 and 25,000 cells/well to wells of 96-wellplate. Umbilicus-derived cells were cultured in Growth Medium ingelatin-coated T75 flasks (Corning, Corning, N.Y.). Umbilicus-derivedcells at passage 18 were transferred to wells at a density of 50,000cells/well. Culture medium was removed from each well aftercentrifugation at 150×g for 5 minutes. Cells were suspended in PBSwithout calcium and magnesium. Cells incubated with anti-tissue factorantibody cells were incubated with 20 micrograms/milliliter CNTO 859(Centocor, Malvern, Pa.) for 30 minutes. Calcium chloride (30microliters) was added to each well. The plate was promptly placed in atemperature-controlled microplate reader and absorbance was measured at405 nanometers at 40 second intervals for 30 minutes.

Antibody Staining. Cells were washed in PBS and detached from the flaskwith Trypsin/EDTA (Gibco, Carlsbad, Calif.). Cells were harvested,centrifuged, and resuspended in 3% (v/v) FBS in PBS at a cellconcentration of 1×10⁷ per milliliter. Antibody was added to 100microliters of cell suspension according to the manufacturer'sspecifications. The cells were incubated in the dark for 30 minutes at4° C. After incubation, cells were washed with PBS, then centrifuged at150×g for 5 minutes to remove unbound antibody. Cells were resuspendedin 100 microliters of 3% FBS and secondary antibody added in accordancewith the manufacturer's instructions. Cells were incubated in the darkfor 30 minutes at 4° C. After incubation, cells were washed with PBS andcentrifuged to remove unbound secondary antibody. Washed cells wereresuspended in 500 microliters of PBS and analyzed via flow cytometry.

Flow Cytometry Analysis. Flow cytometry analysis was performed with aFACSCalibur instrument (Becton Dickinson, San Jose, Calif.).

Results

Flow cytometry analysis revealed that umbilicus-derived postpartum cellsare less active in promoting plasma clotting than the J82 cells.Although a plasma clotting assay demonstrated that the tissue factorpresent in the umbilicus-derived cells was active, clotting took longerthan with the J-82 cells, as evidenced by the longer time tohalf-maximal absorbance (T/2 to max; Table 11-1). The T ½ to max isinversely proportional to the number of J-82 cells. Umbilicus-derivedcells decreased the clotting rate as indicated by the T ½ to max.Clotting was observed with both early (P5) and late (P18) passagedcells. Preincubation of umbilical cells with CNTO 859, an antibody totissue factor, inhibited the clotting reaction, establishing that tissuefactor was responsible for the clotting. TABLE 11-1 The effect of humantissue factor (Simplastin ®) and umbilicus- derived cells (Umb) onplasma clotting. The time to half maximal absorbance (T½ to max) at theplateau in seconds was used as a measurement unit. T½ to max (seconds)Standard (Simplastin ® Dilution) 1:2 61 1:4 107 1:8 147  1:16 174  1:32266  1:64 317  1:128 378    0 1188 (negative control) J-82 cells100,000  122 50,000 172 25,000 275 Umb P5 50,000 833 Umb P18 50,000 443

Summary. Umbilicus-derived PPDCs produce some tissue factor, but theaddition of an antibody against tissue factor can inhibit the clottingactivity of the tissue factor. Tissue factor is normally found on cellsin a conformation that is inactive, but which is activated by mechanicalor chemical (e.g., LPS) stress (Sakariassen et al. (2001) Thromb. Res.104:149-74; Engstad et al. (2002) Int. Immunopharmacol. 2:1585-97).Thus, minimization of stress during the preparation process of PPDCs mayprevent activation of tissue factor. In addition to the thrombogenicactivity, tissue factor has been associated with angiogenic activity.For this reason, tissue factor activity may be beneficial whenumbilicus-derived PPDCs are transplanted in tissue, but should beinhibited when PPDCs are injected intravenously.

EXAMPLE 12 Transplantation of Umbilicus-Derived Cells

Cells derived from the postpartum umbilical cord are useful forregenerative therapies. Tissue produced by SCID mice followingtransplantation of a biodegradable material with and withoutumbilicus-derived cells was evaluated. The materials evaluated wereVICRYL nonwoven scaffolds, 35/65 PCL/PGA foam, and a self-assemblingpeptide hydrogel.

Methods & Materials

Cell Culture. Umbilicus-derived cells were grown in Growth Medium ingelatin-coated flasks.

Matrix Preparation. A nonwoven scaffold was prepared using a traditionalneedle punching technique as described below. Fibers, comprised of asynthetic absorbable copolymer of glycolic and lactic acids (PGA/PLA),sold under the tradename VICRYL were obtained from Ethicon, Inc.(Somerville, N.J.). The fibers were filaments of approximately 20microns in diameter. The fibers were cut and crimped into substantiallyuniform 2-inch lengths to form 2-inch staple fibers. A dry layneedle-punched nonwoven matrix was prepared utilizing the VICRYL staplefibers. The staple fibers were opened and carded on standard nonwovenmachinery. The resulting mat was in the form of webbed staple fibers.The webbed staple fibers were needle-punched to form the dry layneedle-punched nonwoven scaffold. The nonwoven scaffold was rinsed inwater followed by another incubation in ethanol to remove any residualchemicals or processing aids used during the manufacturing process.

Foams, composed of 35/65 poly(epsilon-caprolactone)/poly(glycolic acid)(35/65 PCL/PGA) copolymer, were formed by the process of lyophilization,as described in U.S. Pat. No. 6,355,699.

A self assembling peptide hydrogel (RAD16 self-assembling peptides (3DMatrix, Cambridge, Mass.)) was obtained as a sterile 1% (w/v) solutionin water.

Sample Preparation. One million viable cells were seeded in 15microliters Growth Medium onto 5 millimeter diameter, 2.25 millimeterthick VICRYL nonwoven scaffolds (64.33 milligrams/cc); or 5 millimeterdiameter 35/65 PCL/PGA foam disks. Cells were allowed to attach for twohours before adding more Growth Medium to cover the scaffolds. Cellswere grown on scaffolds overnight. Control scaffolds without cells werealso incubated in medium.

The self-assembling peptide solution was mixed 1:1 with 1×10⁶ cells in10% (w/v) sucrose (Sigma, St Louis, Mo.), 10 millimolar HEPES (pH about7), in Dulbecco's modified medium (DMEM; Gibco) immediately before use.The final concentration of cells in the self-assembling peptide hydrogelwas 1×10⁶ cells/100 microliters.

Test Material (N=4/condition)

1. VICRYL nonwoven+1×10⁶ umbilicus-derived cells

2. 35/65 PCL/PGA foam+1×10⁶ umbilicus-derived cells

3. Self-assembling peptide+1×10⁶ umbilicus-derived cells

4. 35/65 PCL/PGA foam

5. VICRYL nonwoven

Animal Preparation. The animals utilized in this study were handled andmaintained in accordance with the current requirements of the AnimalWelfare Act. Compliance with the above Public Laws were accomplished byadhering to the Animal Welfare regulations (9 C.F.R.) and conforming tothe current standards promulgated in the Guide for the Care and Use ofLaboratory Animals, 7th edition.

Animals: Male mice (Mus musculus) (Fox Chase SCID; Harlan SpragueDawley, Inc., Indianapolis, Ind.), were used at 5 weeks of age. Allhandling of the SCID mice took place under a hood. Each animal wasindividually weighed, and anesthetized with an intraperitoneal injectionof a mixture of 60 milligram/kilogram KETASET (ketamine hydrochloride)(Aveco Co., Inc., Fort Dodge, Iowa), 10 milligram/kilogram ROMPUN(xylazine) (Mobay Corp., Shawnee, Kans.) and saline. After induction ofanesthesia, the back of the animal from the dorsal cervical area to thedorsal lumbosacral area was clipped free of hair using electric animalclippers. The area was then scrubbed with chlorhexidine diacetate,rinsed with alcohol, dried, and painted with an aqueous iodophorsolution of 1% available iodine. Ophthalmic ointment was applied to theeyes to prevent drying of the tissue during the anesthetic period.

Subcutaneous Implantation Technique. Four skin incisions, eachapproximately 1 centimeter in length, were made on the dorsa of themice. Two cranial implantation sites, one each to the left and right ofthe vertebral column, were located transversely over the dorsal lateralthoracic region, about 5 millimeters caudal to the palpated inferioredge of the scapula. Two additional implants, one on each side of themidline, were placed transversely over the gluteal muscle area at thecaudal sacro-lumbar level, about 5 millimeters caudal to the palpatediliac crest. Implants were randomly placed in these sites. The skin wasseparated from the underlying connective tissue to make a small pocketand the implant placed (or injected in the case of the self-assemblingpeptide) about 1 centimeter caudal to the incision. The appropriate testmaterial was implanted into the subcutaneous space. The skin incisionwas closed with metal clips.

Animal Housing. Animals were individually housed in microisolator cagesthroughout the course of the study within a temperature range of 64°F.-79° F. and relative humidity of 30% to 70%, and maintained on a 12hour light/12 hour dark (approximately) cycle. The diet consisted ofIrradiated Pico Mouse Chow 5058 (Purina Co.) and water provided adlibitum.

Mice were euthanized by carbon dioxide inhalation at designatedintervals. The subcutaneous implants with the overlying skin wereexcised and frozen for histology.

Histology. Excised skin with implant was fixed with 10% neutral bufferedformalin (Richard-Allan Scientific, Kalamazoo, Mich.). Samples withoverlying and adjacent tissue were centrally bisected,paraffin-processed, and embedded on cut surface using routine methods.Embedded tissue was sectioned (five-micron sections) on a microtome andstained with hematoxylin and eosin (Poly Scientific, Bay Shore, N.Y.)using routine methods.

Results

There was minimal ingrowth of tissue into control foams withoutumbilicus-derived cells implanted subcutaneously in SCID mice after 30days. In contrast, there was extensive tissue fill into foams implantedwith umbilicus-derived cells.

There was some tissue ingrowth in VICRYL nonwoven scaffolds. Nonwovenscaffolds seeded with umbilicus-derived cells showed increased matrixdeposition and mature blood vessels.

Summary. Human umbilicus-derived cells were shown to dramaticallyincrease good quality tissue formation in biodegradable scaffolds.Synthetic absorbable nonwoven scaffolds, foam discs (5.0 millimetersdiameter×1.0 millimeter thick), or self-assembling peptide hydrogelswere seeded with cells derived from human umbilical cord and implantedsubcutaneously bilaterally in the dorsal spine region of SCID mice. Theumbilicus-derived cells enhanced tissue ingrowth and blood vesselformation on the scaffolds in immune deficient mice, compared to that onscaffolds not seeded with umbilicus-derived cells.

EXAMPLE 13 Transplantation of Umbilicus-derived Cells Under the KidneyCapsule

Transplantation of pancreatic islets to the kidney capsule is routinelyperformed to evaluate transplantation methodologies for the treatment ofdiabetes (Refaie et al., 1998). In addition to pancreatic islets, othercells may be differentiated into insulin secreting cells capable ofblood glucose homeostasis. The suitability of umbilicus-derived cellsfor this purpose was evaluated.

Methods & Materials

Cell Culture. Umbilicus-derived cells (isolate 1, P10) were removed fromliquid nitrogen storage and grown in Growth Medium on gelatin(Sigma)-coated T225 (Corning, Corning, N.Y.) flasks until confluent.

The culture medium on umbilicus-derived cells was replaced with Ham'sF12 medium (Gibco) containing 10 millimolar nicatinamide (Sigma), 25millimolar glucose (Sigma), 10 nanogram/milliliter EGF (PeproTech, RockyHill, N.J.), 20 nanogram/milliliter bFGF (PeproTech) and 15 millimolarGLP-1 (Sigma) and cells were further cultured for 2 weeks.

Cells from two flasks were washed with phosphate buffered saline (PBS),and a single cell suspension was obtained by using Trypsin/EDTA (Gibco).Cryopreserved GM-CSF mobilized CD34+ cells were purchased from Cambrex,Walkersville, Md. (lot 1F0174 donor 7956). CD34+ cells were thawed andwashed in DMEM medium.

The cell suspension was washed twice in DMEM. Cell number and viabilitywas estimated after trypan blue (Sigma) staining using a hemocytometer.Aliquots of the cell suspension containing ˜300,000 viable cells werecentrifuged at 150×g and the cells resuspended in approximately 6microliters of DMEM and drawn into a 20 microliter pipette tip connectedto a 1 milliliter syringe. The tip of the pipette tip containing thecells was clamped using a small Ligaclip (Ethicon Endosurgery,Cincinnati Ohio).

Animal preparation. Mice (Mus musculus)/Fox Chase SCID/Male (HarlanSprague Dawley, Inc., Indianapolis, Ind.) were 8 weeks of age. Allhandling of the SCID mice took place under a hood. The mice wereindividually weighed and anesthetized with an intraperitoneal injectionof a mixture of 60 milligram/kilogram KETASET (ketamine hydrochloride,Aveco Co., Inc., Fort Dodge, Iowa) and 10 milligram/kilogram ROMPUN(xylazine, Mobay Corp., Shawnee, Kans.) and saline. After induction ofanesthesia, the entire back of the animal from the dorsal cervical areato the dorsal lumbosacral area was clipped free of hair using electricclippers. The area was scrubbed with chlorhexidine diacetate, rinsedwith alcohol, dried, and painted with an aqueous iodophor solution of 1%available iodine. Ophthalmic ointment was applied to the eyes to preventdrying of the tissue during the anesthetic period. The anesthetized andsurgically prepared animal was placed in the desired recumbent position.A transverse incision was made on the left abdominal side approximately2 centimeters caudal to the rib cage of animal. The kidney was exposedand the capsule pierced with a 26-gauge needle. A capsule lance(modified glass pipette tip) was used to create a space beneath thekidney capsule into which the cells were introduced. The cells wereinjected via a syringe with a micropipette tip attached. The pocket wasclosed by passing an ophthalmic cautery pen (Aaron Medical Industries,St. Petersburg, Fla.) over the opening (not touching the kidney). Thekidney was placed back in the correct anatomical position, and themuscle layer sutured closed. The skin was closed with wound clips.

The experimental design comprised one cell transplantation in eachmouse; four treatments with n-value of 4 per treatment; and threetime-points (1, 14 & 30 days).

Mice were euthanized by carbon dioxide inhalation at their designatedintervals. The kidney implantation sites were excised and frozen forhistology.

Immunohistochemistry. Frozen kidney implantation site were embedded onedge in O.C.T. Compound (Sakura Inc., Torrance, Calif.). The kidneytissue was trimmed by cryosectioning to yield a five-micron section ofthe implantation site and adjacent tissue. Yielded sections were fixedin freshly prepared 4% paraformaldehyde (EM Sciences Gibbstown, N.J.) inphosphate buffered saline (Gibco) for 15 minutes. Sections were washedin PBS and incubated in 3% goat serum in PBS blocking solution for onehour. Blocking solution was removed by gentle aspiration. Sections wereincubated in anti-human nuclei antibody (Chemicon International,Temecula, Calif.) diluted 1:100 in blocking solution for one hour.Sections were washed with PBS and incubated in florescent labeled goatanti-mouse IgG antibody (Molecular Probes Eugene, Oreg.), diluted 1:200in blocking solution for 30 minutes in absence of light. Sections werewashed in PBS and incubated in 10 micromolar DAPI (Molecular Probes) forfive minutes. Sections were washed in PBS and examined by fluorescentmicroscopy.

Tri-Chrome Staining. Frozen kidney implantation sites were embedded onedge in O.C.T. Compound (Sakura Inc.). The kidney tissue was trimmed bycryosectioning to yield a five-micron section of the implantation siteand adjacent tissue. Yielded sections were fixed in 10% neutral bufferedformalin (Richard-Allan Scientific) for 15 minutes. Sections werestained tri-chrome (Poly Scientific) using manufactures methods.

Treatments:

1. 3×10³ cells from umbilical cord

2. 3×10³ cells from umbilical cord+3×10³ CD34⁺ cells

Added three animals as control (No cells)

Results

The viability of the umbilicus-derived cells was ˜75%; that of the CD34⁺cells was 95%. Initial attempts to transplant 1×10⁶ viable cells wereunsuccessful due to the kidney capsule not being large enough toaccommodate the cells. Cells were transplanted within 3 hours oftrypsinization. The localization of postpartum cells under the kidneycapsule was observed microscopically. There were no apparent differencesin the number and distribution of umbilicus-derived cells with orwithout CD34⁺ cells at each time point. There was an apparent decreasein cell numbers over time.

Staining of cells under the kidney capsule showed the retention oftransplanted cells. Human cells were detected using the human nuclearantigen. All cells (human and mouse) were detected using DAPI.

Umbilicus-derived cells were microscopically observed 14 and 30 dayspost-transplantation. Human cells were again stained for human nuclearantigen. TriChrome staining was used to detect the presence of collagen

Summary. Transplantation of cells into the renal capsule was successful.The observed reduction in cell number over time in this experiment maybe due to a number of factors such as the viability of cells at the timeof transplant, innate immunity and insufficient nutrient availabilitydue to vascularization issues. While long-term survival or even growthof the cells in vivo might be useful for certain purposes, it is notrequired for the cells to be used in many applications, nor do theseresults reflect the ability of the cells to survive and grow over longerterms.

REFERENCE

Refaie A., Gabr M. et al., (1998) Experimental islet celltransplantation in rats: Optimization of the transplantation site.Trans. Proc. 30:400-403

EXAMPLE 14

Short-Term Neural Differentiation of Umbilicus-Derived Cells

The ability umbilicus-derived postpartum cells (PPDCs) to differentiateinto neural lineage cells was examined.

Materials & Methods

Isolation and Expansion of Cells. Umbilicus-derived PPDCs were isolatedand expanded as described in Example 1 and 2.

Modified Woodbury-Black Protocol.

(A) This assay was adapted from an assay originally performed to testthe neural induction potential of bone marrow stromal cells (1).Umbilicus PPDCs (P4) were thawed and culture expanded in Growth Media at5,000 cells/cm² until sub-confluence (75%) was reached. Cells were thentrypsinized and seeded at 6,000 cells per well of a Titretek II glassslide (VWR International, Bristol, Conn.). As controls, mesenchymal stemcells (P3; 1F2155; Cambrex, Walkersville, Md.), osteoblasts (P5; CC2538;Cambrex), omental cells (P6), adipose-derived cells (U.S. Pat. No.6,555,374 B1) (P6) and neonatal human dermal fibroblasts (P6; CC2509;Cambrex) were also seeded under the same conditions.

All cells were initially expanded for 4 days in DMEM/F12 medium(Invitrogen, Carlsbad, Calif.) containing 15% (v/v) fetal bovine serum(FBS; Hyclone, Logan, Utah), basic fibroblast growth factor (bFGF; 20nanogram/milliliter; Peprotech, Rocky Hill, N.J.), epidermal growthfactor (EGF; 20 nanogram/milliliter; Peprotech), penicillin (50Units/milliliter), and streptomycin (50 mirograms/milliliter(Invitrogen). After 4 days, cells were rinsed in phosphate-bufferedsaline (PBS; Invitrogen) and were subsequently cultured in DMEM/F12medium+20% (v/v) FBS+penicillin (50 Units/milliliter)+streptomycin (50micrograms/milliliter (Invitrogen) for 24 hours. After 24 hours, cellswere rinsed with PBS. Cells were then cultured for 1-6 hours in aninduction medium which was comprised of DMEM/F12 (serum-free) containing200 millimolar butylated hydroxyanisole, 10 micromolar potassiumchloride, 5 milligram/milliliter insulin, 10 micromolar forskolin, 4micromolar valproic acid, and 2 micromolar hydrocortisone (all chemicalsfrom Sigma, St. Louis, Mo.). Cells were then fixed in cold (−20° C.)100% methanol and immunocytochemistry was performed (see methods below)to assess human nestin protein expression.

(B) PPDCs (umbilicus, P11) and adult human dermal fibroblasts (1F1853,P11) were thawed and culture expanded in Growth Medium at 5,000cells/cm² until sub-confluence (75%) was reached. Cells were thentrypsinized and seeded at similar density as in (A), but onto (1) 24well tissue culture-treated plates (TCP, Falcon brand, VWRInternational), (2) TCP wells+2% (w/v) gelatin adsorbed for 1 hour atroom temperature, or (3) TCP wells+20 micrograms/milliliter adsorbedmouse laminin (adsorbed for a minimum of 2 hours at 37° C.; Invitrogen).

As in (A) above, cells were initially expanded and media switched at theaforementioned timeframes. One set of cultures was fixed at 5 days andsix hours with cold (4° C.) 4% (w/v) paraformaldehyde (Sigma) for 10minutes at room temperature. In the second set of cultures, the mediumwas removed and switched to Neural Progenitor Expansion medium (NPE)consisting of Neurobasal-A medium (Invitrogen) containing B27 (B27supplement; Invitrogen), L-glutamine (4 millimolar), penicillin (50Units/milliliter), and streptomycin (50 mirograms/milliliter(Invitrogen). NPE medium was further supplemented with retinoic acid(RA; 1 micromolar; Sigma). This medium was removed 4 days later andcultures were fixed with cold (4° C.) 4% (w/v) paraformaldehyde (Sigma)for 10 minutes at room temperature, and stained for nestin, GFAP, andTuJ1 protein expression (see Table 14-1). TABLE 14-1 Summary of PrimaryAntibodies Used Antibody Concentration Vendor Rat 401 (nestin) 1:200Chemicon, Temecula, CA Human Nestin 1:100 Chemicon TuJ1 (BIII Tubulin)1:500 Sigma, St. Louis, MO GFAP  1:2000 DakoCytomation, Carpinteria, CATyrosine hydroxylase (TH)  1:1000 Chemicon GABA 1:400 Chemicon Desmin(mouse) 1:300 Chemicon Alpha smooth muscle actin 1:400 Sigma Humannuclear protein 1:150 Chemicon (hNuc)

Two Stage Differentiation Protocol. Umbilicus-derived PPDCs (P11), adulthuman dermal fibroblasts (P11; 1F1853; Cambrex) were thawed and cultureexpanded in Growth Medium at 5,000 cells/cm until sub-confluence (75%)was reached. Cells were then trypsinized and seeded at 2,000 cells/cm²,but onto 24 well plates coated with laminin (BD Biosciences, FranklinLakes, N.J.) in the presence of NPE media supplemented with bFGF (20nanograms/milliliter; Peprotech, Rocky Hill, N.J.) and EGF (20nanograms/milliliter; Peprotech) (whole media composition furtherreferred to as NPE+F+E). At the same time, adult rat neural progenitorsisolated from hippocampus (P4; (062603)) were also plated onto 24 welllaminin-coated plates in NPE+F+E media. All cultures were maintained insuch conditions for a period of 6 days (cells were fed once during thattime) at which time media were switched to the differentiationconditions listed in Table 14-2 for an additional period of 7 days.Cultures were fixed with ice-cold (4° C.) 4% (w/v) paraformaldehyde(Sigma) for 10 minutes at room temperature, and stained for human or ratnestin, GFAP, and TuJ1 protein expression. TABLE 14-2 Summary ofConditions for Two-Stage Differentiation Protocol A B COND. #PRE-DIFFERENTIATION 2^(nd) STAGE DIFF 1 NPE + F (20 ng/ml) + E (20ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml) 2 NPE + F (20 ng/ml) + E(20 ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml) + RA (1 micromolar) 3NPE + F (20 ng/ml) + E (20 ng/ml) NPE + RA (1 micromolar) 4 NPE + F (20ng/ml) + E (20 ng/ml) NPE + F (20 ng/ml) + E (20 ng/ml) 5 NPE + F (20ng/ml) + E (20 ng/ml) Growth Medium 6 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + rhGDF-5 (20 ng/ml) 7 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + BMP7 (20 ng/ml) 8 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + GDNF (20 ng/ml) 9 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + rhGDF-5 (20 ng/ml) 10 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + BMP7 (20 ng/ml) 11 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + GDNF (20 ng/ml) 12 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + rhGDF-5 (20 ng/ml) 13 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + BMP7 (20 ng/ml) 14 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + GDNF (20 ng/ml) 15 NPE + F (20 ng/ml) + E (20 ng/ml)NPE + rhGDF-5 (20 ng/ml) 16 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + BMP7(20 ng/ml) 17 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + GDNF (20 ng/ml)

Multiple Growth Factor Induction Protocol. Umbilicus-derived PPDCs (P11)were thawed and culture expanded in Growth Medium at 5,000 cells/cm²until sub-confluence (75%) was reached. Cells were then trypsinized andseeded at 2,000 cells/cm², onto 24 well laminin-coated plates (BDBiosciences) in the presence of NPE+F (20/milliliter)+E (20nanograms/milliliter). In addition, some wells contained NPE+F+E+2% FBSor 10% FBS. After four days of “pre-differentiation” conditions, allmedia were removed and samples were switched to NPE medium supplementedwith sonic hedgehog (SHH; 200 nanograms/milliliter; Sigma, St. Louis,Mo.), FGF8 (100 nanograms/milliliter; Peprotech), BDNF (40nanograms/milliliter; Sigma), GDNF (20 nanograms/milliliter; Sigma), andretinoic acid (1 micromolar; Sigma). Seven days post medium change,cultures were fixed with ice-cold (4° C.) 4% (w/v) paraformaldehyde(Sigma) for 10 minutes at room temperature, and stained for humannestin, GFAP, TuJ1, desmin, and alpha-smooth muscle actin expression.

Neural Progenitor Co-Culture Protocol. Adult rat hippocampal progenitors(062603) were plated as neurospheres or single cells (10,000 cells/well)onto laminin-coated 24 well dishes (BD Biosciences) in NPE+F (20nanograms/milliliter)+E (20 nanograms/milliliter).

Umbilicus-derived PPDCs (P11) were thawed and culture expanded in NPE+F(20 nanograms/milliliter)+E (20 nanograms/milliliter) at 5,000 cells/cm²for a period of 48 hours. Cells were then trypsinized and seeded at2,500 cells/well onto existing cultures of neural progenitors. Theexisting medium was exchanged for fresh medium. Four days later,cultures were fixed with ice-cold (4° C.) 4% (w/v) paraformaldehyde(Sigma) for 10 minutes at room temperature, and stained for humannuclear protein (hNuc, Chemicon) (Table 14-1 above) to identify PPDCs.

Immunocytochemistry. Immunocytochemistry was performed using theantibodies listed in Table 14-1. Cultures were washed withphosphate-buffered saline (PBS) and exposed to a protein blockingsolution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula,Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 30 minutes toaccess intracellular antigens. Primary antibodies, diluted in blockingsolution, were then applied to the cultures for a period of 1 hour atroom temperature. Primary antibody solutions were removed and cultureswashed with PBS prior to application of secondary antibody solutions (1hour at room temperature) containing blocking solution along with goatanti-mouse IgG-Texas Red (1:250; Molecular Probes, Eugene, Oreg.) andgoat anti-rabbit IgG-Alexa 488 (1:250; Molecular Probes). Cultures werethen washed and 10 micromolar DAPI (Molecular Probes) was applied for 10minutes to help visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). Positive staining representedfluorescence signal above control staining where the entire procedureoutlined above was followed with the exception of application of aprimary antibody solution. Representative images were captured using adigital color videocamera and ImagePro software (Media Cybernetics,Carlsbad, Calif.). For triple-stained samples, each image was takenusing only one emission filter at a time. Layered montages were thenprepared using Adobe Photoshop software (Adobe, San Jose, Calif.).

Results

Woodbury-Black Protocol.

(A) Upon incubation in this neural induction composition, all cell typestransformed into cells with bipolar morphologies and extended processes.Other larger non-bipolar morphologies were also observed. Furthermore,the induced cell populations stained positively for nestin, a marker ofmultipotent neural stem and progenitor cells.

(B) When repeated on tissue culture plastic (TCP) dishes, nestinexpression was not observed unless laminin was pre-adsorbed to theculture surface. To further assess whether nestin-expressing cells couldthen go on to generate mature neurons, PPDCs and fibroblasts wereexposed to NPE+RA (1 micromolar), a medium composition known to inducethe differentiation of neural stem and progenitor cells into such cells(2, 3, 4). Cells were stained for TuJ1, a marker for immature and matureneurons, GFAP, a marker of astrocytes, and nestin, a marker indicativeof neural progenitors. TuJ1 expression was not turned on, nor were cellswith neuronal morphology observed under any of the tested conditions,suggesting that neurons were not generated in the short term.Furthermore, nestin and GFAP expression, which were found in PPDCs andfibroblasts on laminin-coated substrates, were not produced under theseconditions.

Two Stage Differentiation Results. Umbilicus-derived cell isolates, aswell as human fibroblasts and rodent neural progenitors (as negative andpositive control cell types, respectively), were plated on laminin(neural-promoting)-coated dishes and exposed to 13 different growthconditions (and two control conditions) known to promote differentiationof neural progenitors into neurons and astrocytes. In addition, twoconditions were added to examine the influence of GDF5, and BMP7 on PPDCdifferentiation. Generally, a two-step differentiation approach wastaken, where the cells were first placed in neural progenitor expansionconditions for a period of 6 days followed by full differentiationconditions for 7 days. Morphologically, umbilical cells exhibitedfundamental changes in cell morphology throughout the time-course ofthis procedure. However, in no cases were neuronal or astrocytic-shapedcells observed except for in control, neural progenitor-platedconditions. Immunocytochemistry, negative for human nestin, TuJ1, andGFAP confirmed these morphological observations. Results are summarizedin Table 14-3 below. TABLE 14-3 Staining results for human nestin, GFAP,and TuJ1 respectively in Two Stage Differentiation Experiment. Notethat + means that at least a portion (>0%) of the cells were positivefor the stain indicated. Human nestin: immature neural stem andprogenitor cells; GFAP: astrocytes; TuJ1: immature and mature neurons.Umbilical Neural CONDITION Fibroblasts PPDCs Progenitors 1 −/−/− −/−/−+/+/+ 2 −/−/− −/−/− +/+/+ 3 −/−/− −/−/− +/+/+ 4 −/−/− −/−/− +/+/+ 5−/−/− −/−/− +/+/+ 6 −/−/− −/−/− +/+/+ 7 −/−/− −/−/− +/+/+ 8 −/−/− −/−/−+/+/+ 9 −/−/− −/−/− +/+/+ 10 −/−/− −/−/− +/+/+ 11 −/−/− −/−/− +/+/+ 12−/−/− −/−/− +/+/+ 13 −/−/− −/−/− +/+/+ 14 −/−/− −/−/− +/+/+ 15 −/−/−−/−/− +/+/+ 16 −/−/− −/−/− +/+/+ 17 −/−/− −/−/− +/+/+

Multiple Growth Factor Induction Results. Following a one week exposureto a variety of neural differentiation agents, cells were stained formarkers indicative of neural progenitors (human nestin), neurons (TuJ1),and astrocytes (GFAP). Cells grown in the first stage in non-serumcontaining media had different morphologies than those cells in serumcontaining (2% or 10%) media, indicating potential neuraldifferentiation. Specifically, following a two step procedure ofexposing umbilical PPDCs to EGF and bFGF, followed by SHH, FGF8, GDNF,BDNF, and retinoic acid, cells showed long extended processes similar toa morphology of cultured astrocytes. When 2% FBS or 10% FBS wereincluded in the first stage of differentiation, cell number wasincreased and cell morphology was unchanged from control cultures athigh density. Potential neural differentiation was not evidenced byimmunocytochemical analysis for human nestin, TuJ1, or GFAP.

Neural Progenitor and PPDC Co-culture Procedures. Umbilicus-derivedcells were plated onto cultures of rat neural progenitors seeded twodays earlier in neural expansion conditions (NPE+F+E). While visualconfirmation of plated umbilicus proved that these cell were plated assingle cells, human-specific nuclear staining (hNuc) 4 days post-plating(6 days total length of experiment) showed that they tended to ball upand avoid contact with the neural progenitors. Furthermore, whereumbilicus cells attached, these cells spread out and appeared to beinnervated by differentiated neurons that were of rat origin suggestingthat the umbilical cells may have differentiated into muscle cells. Thisobservation was based upon morphology under phase contrast microscopy.Another observation was that typically large cell bodies (larger thanneural progenitors) possessed morphologies resembling neuralprogenitors, with thin processes spanning out in multiple directions.HNuc staining (found in one half of the cell's nucleus) suggested thatin some cases these human cells may have fused with rat progenitors andassumed their phenotype. Controls wells containing neural progenitorsonly had fewer total progenitors and apparent differentiated cells thandid co-culture wells containing umbilicus, further indicating thatumbilicus-derived cells influenced the differentiation and behavior ofneural progenitors either by release of chemokines and cytokines, or bycontact-mediated effects.

Summary. Multiple protocols were conducted to determine the short termpotential of umbilicus-derived PPDCs to differentiate into neurallineage cells. These included phase contrast imaging of morphology incombination with immunocytochemistry for nestin, TuJ1, and GFAP,proteins associated with multipotent neural stem and progenitor cells,immature and mature neurons, and astrocytes, respectively. Evidence wasobserved to suggest that neural differentiation occurred in certaininstances in these short-term protocols.

Several notable observations were made in co-cultures of PPDCs withneural progenitors. This approach, using human PPDCs along with axenogeneic cell type allowed for absolute determination of the origin ofeach cell in these cultures. First, some cells were observed in thesecultures where the cell cytoplasm was enlarged, with neurite-likeprocesses extending away from the cell body, yet only half of the bodylabeled with hNuc protein. Those cells may be human PPDCs that havedifferentiated into neural lineage cells or they may be PPDCs that havefused with neural progenitors of rat origin. Second, it appeared thatneural progenitors extended neurites to PPDCs in a way that indicatesthe progenitors differentiated into neurons and innervated the PPDCs.Third, cultures of neural progenitors and PPDCs had more cells of ratorigin and larger amounts of differentiation than control cultures ofneural progenitors alone, further indicating that plated PPDCs providedsoluble factors and or contact-dependent mechanisms that stimulatedneural progenitor survival, proliferation, and/or differentiation.

REFERENCES

-   (1) Woodbury, D. et al. (2000). J Neurosci. Research. 61(4): 364-70.-   (2) Jang, Y. K. et al. (2004). J. Neurosci. Research. 75(4): 573-84.-   (3) Jones-Villeneuve, E. M. et al. (1983). Mol Cel Biol. 3(12):    2271-9.-   (4) Mayer-Proschel, M. et al. (1997). Neuron. 19(4): 773-85.

EXAMPLE 15 Long-Term Neural Differentiation of Umbilicus-Derived Cells

The ability of umbilicus-derived cells to undergo long-termdifferentiation into neural lineage cells was evaluated.

Materials & Methods

Isolation and Expansion of Postpartum Cells (PPDCs). Umbilicus-derivedPPDCs were isolated and expanded as described in Examples 1 and 2.

Umbilicus-derived Cell Thaw and Plating. Frozen aliquots ofumbilicus-derived cells P11 and P12, previously grown in Growth Mediumwere thawed and plated at 5,000 cells/cm² in T-75 flasks coated withlaminin (BD, Franklin Lakes, N.J.) in Neurobasal-A medium (Invitrogen,Carlsbad, Calif.) containing B27 (B27 supplement, Invitrogen),L-glutamine (4 millimolar), penicillin (50 Units/milliliter), andstreptomycin (50 micrograms/milliliters), the combination of which isherein referred to as Neural Progenitor Expansion (NPE) media. NPEmedium was further supplemented with bFGF (20 nanograms/milliliter,Peprotech, Rocky Hill, N.J.) and EGF (20 nanograms/milliliter,Peprotech, Rocky Hill, N.J.), herein referred to as NPE+bFGF+EGF.

Control Cell Plating. In addition, adult human dermal fibroblasts (P11,Cambrex, Walkersville, Md.) and mesenchymal stem cells (P5, Cambrex)were thawed and plated at the same cell seeding density onlaminin-coated T-75 flasks in NPE+bFGF+EGF. As a further control,fibroblasts and umbilical cells were grown in Growth Medium for theperiod specified for all cultures.

Cell Expansion. Media from all cultures were replaced with fresh mediaonce a week and cells observed for expansion. In general, each culturewas passaged one time over a period of one month because of limitedgrowth in NPE+bFGF+EGF.

Immunocytochemistry. After a period of one month, all flasks were fixedwith cold (4° C.) 4% (w/v) paraformaldehyde (Sigma) for 10 minutes atroom temperature. Immunocytochemistry was performed using antibodiesdirected against TuJ1 (BIII Tubulin; 1:500; Sigma, St. Louis, Mo.) andGFAP (glial fibrillary acidic protein; 1:2000; DakoCytomation,Carpinteria, Calif.). Briefly, cultures were washed withphosphate-buffered saline (PBS) and exposed to a protein blockingsolution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula,Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 30 minutes toaccess intracellular antigens. Primary antibodies, diluted in blockingsolution, were then applied to the cultures for a period of 1 hour atroom temperature. Primary antibody solutions were then removed and thecultures were washed with PBS prior to application of secondary antibodysolutions (1 hour at room temperature) containing block along with goatanti-mouse IgG-Texas Red (1:250; Molecular Probes, Eugene, Oreg.) andgoat anti-rabbit IgG-Alexa 488 (1:250; Molecular Probes). Cultures werethen washed and 10 micromolar DAPI (Molecular Probes) applied for 10minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Results

NPE+bFGF+EGF Media Slows Proliferation of PPDCs and Alters TheirMorphology. Immediately following plating, a subset of umbilical cellsattached to the culture flasks coated with laminin. This may have beendue to cell death as a function of the freeze/thaw process or because ofthe new growth conditions. Cells that did attach adopted morphologiesdifferent than those observed in Growth Media.

Upon confluence, cultures were passaged and observed for growth. Verylittle expansion took place of those cells that survived passage. Atthis point, very small cells with no spread morphology and withphase-bright characteristics began to appear in cultures of umbilicalcells. These areas of the flask were followed over time. From thesesmall cells, bifurcating processes emerged with varicosities along theirlengths, features very similar to previously described PSA-NCAM+neuronalprogenitors and TuJ1+immature neurons derived from brain and spinal cord(1, 2). With time, these cells became more numerous, yet still were onlyfound in clones.

Clones of Umbilical Cells Express Neuronal, but not Glial Proteins.Cultures were fixed at one month post-thawing/plating and stained forthe neuronal protein TuJ1 and GFAP, an intermediate filament found inastrocytes. While all control cultures grown in Growth Medium and humanfibroblasts and MSCs grown in NPE+bFGF+EGF medium were found to beTuJ1-/GFAP-, umbilical cells turned on expression of TuJ1. Expressionwas observed in cells with and without neuronal-like morphologies. Noexpression of GFAP was observed in either culture. The percentage ofcells expressing TuJ1 with neuronal-like morphologies was less than orequal to 1% of the total population (n=3 umbilical isolates tested).

Summary. Methods for generating differentiated neurons (based on TuJ1expression AND neuronal morphology) from umbilical cells were developed.While expression for TuJ1 was not examined earlier than one month invitro, it is clear that at least a small population of umbilicus-derivedcells can give rise to neurons either through default differentiation orthrough long-term induction following one month's exposure to a minimalmedia supplemented with L-glutamine, basic FGF, and EGF.

REFERENCES FOR EXAMPLE 15

-   (1) Mayer-Proschel, M. et al. (1997). Neuron. 19(4): 773-85.-   (2) Yang, H. et al. (2000). PNAS. 97(24): 13366-71.

EXAMPLE 16 Umbilical Derived Cell Trophic Factors for Neural ProgenitorDifferentiation

The influence of umbilicus-derived postpartum cells (PPDCs) on adultneural stem and progenitor cell survival and differentiation throughnon-contact dependent (trophic) mechanisms was examined.

Materials & Methods

Adult Neural Stem and Progenitor Cell Isolation. Fisher 344 adult ratswere sacrificed by CO₂ asphyxiation followed by cervical dislocation.Whole brains were removed intact using bone rongeurs and hippocampustissue dissected based on coronal incisions posterior to the motor andsomatosensory regions of the brain (1). Tissue was washed inNeurobasal-A medium (Invitrogen, Carlsbad, Calif.) containing B27 (B27supplement; Invitrogen), L-glutamine (4 millimolar; Invitrogen), andpenicillin (50 Units/milliliter) and streptomycin (50micrograms/milliliter) (Invitrogen), the combination of which is hereinreferred to as Neural Progenitor Expansion (NPE) medium. NPE medium wasfurther supplemented with bFGF (20 nanograms/milliliter, Peprotech,Rocky Hill, N.J.) and EGF (20 nanograms/milliliter, Peprotech, RockyHill, N.J.), herein referred to as NPE+bFGF+EGF.

Following wash, the overlying meninges were removed, and the tissueminced with a scalpel. Minced tissue was collected and trypsin/EDTA(Invitrogen) added as 75% of the total volume. DNAse (100 microlitersper 8 milliliters total volume, Sigma, St. Louis, Mo.) was also added.Next, the tissue/medium was sequentially passed through an 18 gaugeneedle, 20 gauge needle, and finally a 25 gauge needle one time each(all needles from Becton Dickinson, Franklin Lakes, N.J.). The mixturewas centrifuged for 3 minutes at 250×g. Supernatant was removed, freshNPE+bFGF+EGF was added and the pellet resuspended. The resultant cellsuspension was passed through a 40 micron cell strainer (BectonDickinson), plated on laminin-coated T-75 flasks (Becton Dickinson) orlow cluster 24-well plates (Becton Dickinson), and grown in NPE+bFGF+EGFmedia until sufficient cell numbers were obtained for the studiesoutlined.

PPDC Cell Plating. Postpartum-derived cells (P12) previously grown inGrowth Media were plated at 5,000 cells/transwell insert (sized for 24well plate) and grown for a period of one week in Growth Media ininserts to achieve confluence.

Adult Neural Progenitor Plating. Neural progenitors, grown asneurospheres or as single cells, were seeded onto laminin-coated 24 wellplates at an approximate density of 2,000 cells/well in NPE+bFGF+EGF fora period of one day to promote cellular attachment. One day later,transwell inserts containing postpartum cells were added according tothe following scheme:

(1) Transwell (umbilicus in Growth Media, 200 microliters)+neuralprogenitors (NPE+bFGF+EGF, 1 milliliter)

(2) Transwell (adult human dermal fibroblasts [IF1853; Cambrex,Walkersville, Md.] P12 in Growth Media, 200 microliters)+neuralprogenitors (NPE+bFGF+EGF, 1 milliliter)

(3) Control: neural progenitors alone (NPE+bFGF+EGF, 1 milliliter)

(4) Control: neural progenitors alone (NPE only, 1 milliliter)

Immunocytochemistry. After 7 days in co-culture, all conditions werefixed with cold 4% (w/v) paraformaldehyde (Sigma) for a period of 10minutes at room temperature. Immunocytochemistry was performed usingantibodies directed against the epitopes listed in Table 16-1. Briefly,cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma) for 30 minutes to access intracellular antigens. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. Next, primaryantibodies solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing blocking solution along with goat anti-mouse IgG-Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG-Alexa488 (1:250; Molecular Probes). Cultures were then washed and 10micromolar DAPI (Molecular Probes) applied for 10 minutes to visualizecell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.). TABLE 16-1 Summary of Primary Antibodies Used AntibodyConcentration Vendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA TuJ1(BIII Tubulin) 1:500 Sigma, St. Louis, MO Tyrosine  1:1000 Chemiconhydroxylase (TH) GABA 1:400 Chemicon GFAP  1:2000 DakoCytomation,Carpinteria, CA Myelin Basic 1:400 Chemicon Protein (MBP)

Quantitative Analysis of Neural Progenitor Differentiation. Hippocampalneural progenitor differentiation was examined and quantified. A minimumof 1,000 cells were counted per condition, or if less, the total numberof cells observed in that condition. The percentage of cells positivefor a given stain was assessed by dividing the number of positive cellsby the total number of cells as determined by DAPI (nuclear) staining.

Mass Spectrometry Analysis & 2D Gel Electrophoresis. In order toidentify unique, secreted factors as a result of co-culture, conditionedmedia samples taken prior to culture fixation were frozen down at −80°C. overnight. Samples were then applied to ultrafiltration spin devices(nominal MW cutoff 30 kD). Retentate was subjected to immunoaffinitychromatography (anti-Human-albumin; IgY) (immunoaffinity did not removealbumin from the samples). Filtrate was analyzed by MALDI-TOFF. Theeluate was applied to Cibachron Blue affinity chromatography. Sampleswere analyzed by SDS-PAGE and 2D gel electrophoresis.

Results

Umbilical Co-culture Stimulates Adult Neural Progenitor Differentiation.Following culture with umbilicus-derived postpartum cells, co-culturedneural progenitor cells derived from adult rat hippocampus exhibitedsignificant differentiation along all three major lineages in thecentral nervous system. This effect was clearly observed after five daysin co-culture, with numerous cells elaborating complex processes andlosing their phase bright features characteristic of dividing progenitorcells. Conversely, neural progenitors grown alone in the absence of bFGFand EGF appeared unhealthy and survival was limited.

After completion of the procedure, cultures were stained for markersindicative of undifferentiated stem and progenitor cells (nestin),immature and mature neurons (TuJ1), astrocytes (GFAP), and matureoligodendrocytes (MBP). Differentiation along all three lineages wasconfirmed while control conditions did not exhibit significantdifferentiation as evidenced by retention of nestin-positive stainingamongst the majority of cells.

The percentage of differentiated neural progenitors following co-culturewith umbilicus-derived PPDCs was quantified (Table 16-2).Umbilicus-derived cells significantly enhanced the number of matureoligodendrocytes (MBP) (24.0% vs 0% in both control conditions).Furthermore, co-culture enhanced the number of GFAP+astrocytes andTuJ1+neurons in culture (47.2% and 8.7% respectively). These resultswere confirmed by nestin staining indicating that progenitor status waslost following co-culture (13.4% vs 71.4% in control condition 3).

Though differentiation also appeared to be influenced by adult humanfibroblasts, such cells were not able to promote the differentiation ofmature oligodendrocytes nor were they able to generate an appreciablequantity of neurons. Though not quantified, fibroblasts did, however,appear to enhance the survival of neural progenitors and their progenysimilar to findings for umbilicus-derived postpartum cells. TABLE 16-2Quantification of progenitor differentiation in control vs transwellco-culture with umbilicus-derived postpartum cells (E = EGF, F = bFGF).F + E/Umb F + E/F + E F + E/removed Antibody [Cond. 1] [Cond. 3] [Cond.4] TuJ1  8.7%  2.3%  3.6% GFAP 47.2% 30.2% 10.9% MBP 23.0%   0%   0%Nestin 13.4% 71.4% 39.4%

Identification of Unique Compounds. Conditioned media from umbilicaltest conditions along with the appropriate controls (NPE media±1.7%serum, media from co-culture with fibroblasts) were examined fordifferences. Potentially unique compounds were identified and excisedfrom their respective 2D gels.

Summary. Results presented in this example indicate that thedifferentiation of adult neural progenitor cells following co-culturewith umbilicus-derived postpartum cells is particularly profound.Specifically, a significant percentage of mature oligodendrocytes weregenerated in co-cultures of umbilical cells. In view of the lack ofcontact between umbilical cells and the neural progenitors, this resultappears to be a function of soluble factors released from the umbilicalcells (trophic effect).

Several other observations were made. First, there were very few cellsin the control condition where EGF and bFGF were removed. Most cellsdied and on average, there were about 100 cells or fewer per well.Second, it is to be expected that there would be very littledifferentiation in the control condition where EGF and bFGF was retainedin the medium throughout, since this is normally an expansion medium.While approximately 70% of the cells were observed to retain theirprogenitor status (nestin+), about 30% were GFAP+(indicative ofastrocytes). This may be due to the fact that such significant expansionoccurred throughout the course of the procedure that contact betweenprogenitors induced this differentiation. Similar findings have beenreported in the literature (2).

REFERENCES FOR EXAMPLE 16

-   (1) Paxinos, G. & Watson, C. (1997). The Rat Brain in Stereotaxic    Coordinates.-   (2) Song, H. et al. (2002). Nature. 417(6884): 29-32.

EXAMPLE 17 The Effect of Trophic Factors on Angiogenesis

Angiogenesis, or the formation of new vasculature, is necessary for thegrowth of new tissue. Induction of angiogenesis is an importanttherapeutic goal in many pathological conditions. The angiogenicactivity of umbilicus-derived cells in in vitro assays was examined. Awell-established method of assessing angiogenic activity involvingseeding endothelial cells onto a culture plate coated with a basementmembrane extract (Nicosia and Ottinetti (1990) In Vitro Cell Dev. Biol.26(2):119-28), was utilized. Treating endothelial cells on such basementmembranes or extracellular matrix material with angiogenic factors willstimulate the cells to form a network that is similar to capillaries.These types of assays are common in vitro assays for testing stimulatorsand inhibitors of blood vessel formation (Ito et al. (1996) Int. J.Cancer 67(1):148-52). The protocols utilized in this example made use ofa co-culture system with the umbilicus-derived cells seeded onto culturewell inserts. These permeable inserts allow for the passive exchange ofmedia components between the endothelial and the umbilicus-derived cellculture media.

Material & Methods

Cell Culture.

Umbilicus-derived cells. Human umbilical cords were received and cellswere isolated as previously described (Example 1). Cells were culturedin Growth Medium on gelatin-coated tissue culture plastic flasks. Thecultures were incubated at 37° C. with 5% CO₂. Cells used forexperiments were between passages 4 and 12.

Actively growing UDCs were trypsinized, counted, and seeded onto 6.5millimeter diameter tissue culture inserts (COSTAR TRANSWELL, CorningInc., Corning, N.Y.) at 15,000 cells per insert. Cells were cultured onthe inserts for 48-72 hours in Growth Medium with 5% CO₂ at 37° C.

Human mesenchymal stem cells (hMSC). hMSCs were purchased from Cambrex(Walkersville, Md.) and cultured in MSCGM (Cambrex). The cultures wereincubated with 5% CO₂ at 37° C.

Actively growing MSCs were trypsinized and counted and seeded onto 6.5millimeter diameter tissue culture inserts (Corning, Corning, N.Y.) at15,000 cells per insert. Cells were cultured on the inserts for 48-72hours in Growth Medium with 5% CO₂ at 37° C.

Human umbilical vein endothelial cells (HUVEC). HUVEC were obtained fromCambrex (Walkersville, Md.). Cells were grown in separate cultures ineither EBM or EGM endothelial cell media (Cambrex). Cells were grown onstandard tissue cultured plastic with 5% CO₂ at 37° C. Cells used in theassay ranged from passages 4 to 10.

Human coronary artery endothelial cells (HCAEC). HCAEC were purchasedfrom Cambrex Incorporated (Walkersville, Md.). These cells were alsomaintained in separate cultures in either the EBM or EGM mediaformulations. Cells were grown on standard tissue cultured plastic with5% CO₂ at 37° C. Cells used for experiments ranged from passages 4 to 8.

Angiogenesis Assays on Extracellular Matrix. Culture plates were coatedwith extracellular matrix material according to manufacturer'sspecifications. Briefly, extracellular matrix material (MATRIGEL, BDDiscovery Labware, Bedford, Mass.) was thawed at 4° C. and approximately250 microliters were distributed onto each well of a chilled 24-wellculture plate (Corning). The plate was then incubated at 37° C. for 30minutes to allow the material to solidify. Actively-growing endothelialcell cultures were trypsinized and counted. Cells were washed twice inGrowth Medium supplemented with only 2% FBS by centrifugation,resuspension, and aspiration of the supernatant. Cells were seeded ontothe coated wells 20,000 cells per well in approximately 0.5 milliliterGrowth Medium supplemented with only 2% (v/v) FBS. Cells were thenincubated for approximately 30 minutes to allow the cells to settle.

Endothelial cell cultures were then treated with either 10 nanomolarhuman bFGF (Peprotech, Rocky Hill, N.J.) or 10 nanomolar human VEGF(Peprotech, Rocky Hill, N.J.) to serve as positive controls forendothelial cell response. Transwell inserts seeded with postpartumcells were added to appropriate wells with Growth Medium supplementedwith only 2% FBS in the insert chamber. Cultures were incubated in 5%CO₂ at 37° C. for approximately 24 hours. The well plate was removedfrom the incubator, and images of the endothelial cell cultures werecollected with an Olympus inverted microscope (Olympus, Melville, N.Y.).

Results

In a co-culture system with umbilicus-derived cells, HUVEC formstructured cell networks. HUVEC cells form limited cell networks inco-culture experiments with hMSC and with 10 nanomolar bFGF. HUVEC cellswithout any treatment showed very little or no network formation. Theseresults suggest that the umbilicus-derived cells release angiogenicfactors that stimulate the HUVEC. Similarly, HCAECs formed cell networksonly in co-culture with umbilicus-derived cells.

Table 17-1 shows quantities of known angiogenic factors released by UDCsin Growth Medium at atmospheric oxygen conditions. UDCs were seeded ontoinserts as described above. The cells were cultured at 37° C. inatmospheric oxygen for 48 hours on the inserts and then switched to a 2%FBS media and returned at 37° C. for 24 hours. Medium was removed,immediately frozen, and stored at −80° C., and analyzed by theSearchLight multiplex ELISA assay (Pierce Chemical Company, Rockford,Ill.). Results shown are the averages of duplicate measurements. Theresults show that UDCs do not release detectable levels ofplatelet-derived growth factor-bb (PDGFbb), heparin-binding epidermalgrowth factor (HB-EGF), or vascular endothelial growth factor (VEGF).The amounts of angiopoietin 2 (ANG2) detected were less than that of theculture medium control with no cells. The umbilicus-derived cellsreleased measurable quantities of tissue inhibitor ofmetallinoprotease-1 (TIMP-1), thrombopoietin (TPO), and hepatocytegrowth factor (HGF). The amounts of keratinocyte growth factor (KGF) andfibroblast growth factor (FGF) were very low and only slightly abovethose for the control medium. TABLE 17-1 Potential angiogenic factorsreleased from UDCs. Umbilicus- derived cells were cultured in 24 hoursin media with 2% FBS in atmospheric oxygen. Medium was removed andassayed by the SearchLight multiplex ELISA assay (Pierce). Results arethe means of a duplicate analysis. Values are concentrations in themedia reported in picograms per milliliter of culture media. TIMP1 ANG2PDGFBB TPO KGF HGF FGF VEGF HBEGF (pg/mL) (pg/mL) (pg/mL) (pg/mL)(pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) UDCs (P4) 81831.7 <9.8 <2.0365.9 14.1 200.2 5.8 <4.0 <1.2 Media alone <9.8 25.1 <2.0 <6.4 <2.0 <3.2<5.4 <4.0 <1.2

Table 17-2 shows levels of known angiogenic factors released by UDCs at5% O₂. UDCs were seeded onto inserts as described above. The cells werecultured in Growth Medium at 5% oxygen for 48 hours on the inserts andthen switched to a 2% FBS medium and returned to 5% O₂ incubation for 24hours. Medium was removed, immediately frozen, and stored at −80° C.,and analyzed by the SearchLight multiplex ELISA assay (Pierce ChemicalCompany, Rockford, Ill.). Results shown are the averages of duplicatemeasurements. The results show for UDCs are comparable to those underatmospheric oxygen conditions. While there are essentially no changes inthe production of ANG2, PDGFBB, FGF, VEGF, and HB-EGF by UDCs, there wasa slight increase apparent in production of TIMP1, KGF, and HGF, and aslight decrease apparent in production of TPO. These apparentdifferences in raw data were not tested for statistical significance.TABLE 17-2 Potential angiogenic factors released from UDCs. Cells werecultured in 24 hours in media with 2% FBS in 5% oxygen. Medium wasremoved and assayed by the SearchLight multiplex ELISA assay (Pierce).Results are the means of a duplicate analysis. Values are concentrationsin the media reported in picograms per milliter of culture media. TIMP1ANG2 PDGFBB TPO KGF HGF FGF VEGF HBEGF (pg/mL) (pg/mL) (pg/mL) (pg/mL)(pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) UDCs (P4) 50244.7 <9.8 <2.0403.3 10.7 156.8 5.7 <4.0 <1.2 Media alone <9.8 25.1 <2.0 <6.4 <2.0 <3.2<5.4 <4.0 <1.2

Summary. The results of the study show that umbilicus-derived cells canstimulate both human umbilical vein and coronary artery endothelialcells to form networks in an in vitro assay of angiogenesis. This effectis similar to that seen with known angiogenic factors in such assaysystems. These results suggest that UDCs are useful for stimulatingangiogenesis in vivo.

EXAMPLE 18 Differentiation of Umbilicus-Derived Cells into Hepatocytes

A variety of conditions were examined to determine a suitablecombination of basic media and growth factors for the differentiation ofumbilicus-derived cells into hepatocytes. HNF-1alpha, ahepatocyte-specific transcription factor, cytoplasmic intermediatefilament proteins, such as keratin 19 (K19), keratin 8 (K8), andcytokeratin 18 (CK18), which are markers of epithelial cells and twoliver-specific secreted proteins, albumin and cytochrome p450 2B6, wereselected as markers for hepatocyte differentiation (Schwartz et al.(2002) J. Clin. Invest. 109(10):1291-1302; Okumoto et al. (2003)Biochem. Biophys. Res. Commun. 304(4):691-695; Chargracui et al. (2003)Blood 101(8): 2973-2982).

Methods & Materials

Umbilicus-derived cells obtained according to method of Example 1, aswell as neonatal or adult Normal Human Dermal Fibroblasts (NHDF), weregrown in Growth Medium in gelatin-coated T75 flasks. Basic FibroblastGrowth Factor (bFGF), Oncostatin M, Hepatocyte Growth Factor (HGF), StemCell Factor (SCF), and Fibroblast Growth Factor 4 (FGF 4) were fromPeproTech Inc. (Rocky Hill, N.J.). Platelet Derived Growth Factor BB(PDGFbb) was from R&D Systems (Minneapolis, Minn.).

The following conditions were tested:

Method 1

Umbilicus-derived cells (P5), neonatal and adult Normal Human DermalFibroblasts (NHDF). Cells were plated at 22.5×10³ cells/cm² on 1%Matrigel (Becton-Dickinson and Co., Franklin Lakes, N.J.) in serum-freemedium (60% (v/v) low glucose DMEM) (DMEM-LG; Gibco, Carlsbad, Calif.),40% (v/v) MCDB-201 (Sigma, St. Louis, Mo.), supplemented with 1×insulin/transferrin/selenium, 4.7 micrograms/milliliter linoleic acid, 1milligram/milliliter bovine serum albumin, 10 nanomolar Dexamethasone,100 micromolar ascorbic acid phosphate (all from Sigma), 50Units/milliliter penicillin, 50 micrograms/milliliter streptomycin(Gibco), 2% (v/v) FCS (Hyclone Laboratories, Logan, Utah), and 10nanograms/milliliter each EGF and PDGFbb). After 8-12 hours, medium wasremoved, cells were washed twice with PBS (Gibco) and cultured in theabove-described medium without EGF and PDGFbb but supplemented with 20nanograms/milliliter HGF and/or 10 nanograms/milliliter FGF-4 (Schwartzet al. (2002) J. Clin. Invest. 109(10):1291-1302).

Method 2

Umbilicus-derived cells (P5), neonatal and adult NHDF. Cells were seededat lower density (22,500 cells/cm ²) in 24-well plates coated withgelatin and grown as described above.

Method 3

Umbilicus-derived cells (P17), Umbilicus-derived cells (P15),Umbilicus-derived cells (P10), adult NHDF. Cells were seeded at higherdensity (50,000 cells/cm²) in 24-well TCP plates and grown in DMEM(Gibco), B27 Supplement (Gibco), penicillin (50 Units/milliliter),streptomycin (50 micrograms/milliliter), 20 nanograms/milliliter HGFand/or 10 nanograms/milliliter FGF-4. Cells were grown in theseconditions for 4 weeks.

Method 4

Umbilicus-derived cells (P4), Umbilicus-derived cells (P9), neonatal andadult NHDF. Cells were seeded at a density of 5,000 cells/cm² T25 flasksin Chang C medium (Irvine Scientific, Santa Ana, Calif.) on eitherfibronectin (PeproTech, Rocky Hill, N.J.) or gelatin (Sigma) and grownfor two passages until confluence. Cells were then seeded at 1,000cells/cm² in 24-well TCP plates and grown as described above until theyreached about 40-60% confluence.

Method 5

Umbilicus-derived cells (P5) and adult NHDF. Cells were plated in24-well plates on gelatin in Growth Medium supplemented with either 1nanogram/milliliter or 10 nanograms/milliliter oncostatin M (Chargracui(2003) Blood 101(8): 2973-2982). Cells were grown in these conditionsfor 4 weeks.

Method 6

Umbilicus-derived cells (P5) and adult NHDF. Cells were plated in24-well plates on gelatin in Growth Medium supplemented with 10nanograms/milliliter bFGF, 10 nanograms/milliliter HGF, 10nanograms/milliliter SCF. Cells were grown in these conditions for 4weeks (Okumoto et al. (2003) Biochem. Biophys. Res. Commun.304(4):691-695.).

Total RNA isolation and quantitative RT-PCR. RNA was extracted fromumbilicus-derived cells and fibroblasts grown as described in eachprotocol. Cells were lysed with 350 microliters buffer RLT containingbeta-mercaptoethanol (Sigma St. Louis, Mo.) according to themanufacturer's instructions (RNeasy Mini Kit, Qiagen, Valencia, Calif.)and RNA extracted according to the manufacturer's instructions (RNeasyMini Kit, Qiagen, Valencia, Calif.) with a 2.7 Units/sample DNasetreatment (Sigma). RNA was eluted with 50 microliters DEPC-treated waterand stored at −80° C. RNA was reverse transcribed using random hexamerswith the TaqMan reverse transcription reagents (Applied Biosystems,Foster City, Calif.) at 25° C. for 10 minutes, 37° C. for 60 minutes,and 95° C. for 10 minutes. Samples were stored at −20° C.

Real-time PCR. PCR was performed on cDNA samples using ASSAYS-ON-DEMANDgene expression products for albumin (Hs00609411), cytochrome p450 2B6(Hs00167937), GAPDH (Applied Biosystems, Foster City, Calif.) and TaqManUniversal PCR master mix according to the manufacturer's instructions(Applied Biosystems, Foster City, Calif.) using a 7000 sequencedetection system with ABI prism 7000 SDS software (Applied Biosystems,Foster City, Calif.). Thermal cycle conditions were initially 50° C. for2 minutes and 95° C. for 10 minutes followed by 40 cycles of 95° C. for15 seconds and 60° C. for 1 minute. PCR data were analyzed according tomanufacturer's specifications (User Bulletin #2 from Applied Biosystemsfor ABI Prism 7700 Sequence Detection System).

Immunofluorescence. Cell cultures were fixed with cold (4° C.) 4% (w/v)paraformaldehyde for a period of 10 minutes at room temperature.Immunocytochemistry was performed using antibodies directed against thefollowing epitopes: keratin 9 (K9; 1:400; Chemicon, Temecula, Calif.),keratin 19 (K19; 1:400; Chemicon), cytokeratin 18 (CK18; 1:400; Sigma,St. Louis, Mo.), vimentin (1:500; Sigma), desmin (1:150; Sigma), albumin(1:200; Sigma), c-met (1:400; Santa Cruz Biotech, Santa Cruz, Calif.),and HNF-1alpha (1:400; Santa Cruz Biotech). In general, cultures werewashed with phosphate-buffered saline (PBS) and exposed to a proteinblocking solution containing PBS, 4% (v/v) goat serum (Chemicon,Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100, Sigma) for 30minutes to access intracellular antigens. In instances where the epitopeof interest would be located on the cell surface (e.g. c-met), Tritonwas omitted in all steps of the procedure in order to prevent epitopeloss. Primary antibodies, diluted in blocking solution, were thenapplied to the cultures for a period of 1 hour at room temperature.Primary antibody solutions were then removed and cultures washed withPBS prior to application of secondary antibody solutions (1 hour at roomtemperature) containing blocking solution along with goat anti-mouseIgG-Texas Red (1:250; Molecular Probes, Eugene, Oreg.) for K8, K19,CK18, vimentin, and albumin, goat anti-rabbit IgG-Alexa 488 (1:250;Molecular Probes) for desmin and c-met, or donkey anti-goat IgG-FITC(1:150; Santa Cruz Biotech) for HNF-1alpha staining. Cultures werewashed and 10 micromolar DAPI (Molecular Probes) applied for 10 minutesto visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). Representative images werecaptured using a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter. Layered montages were thenprepared using Adobe Photoshop software (Adobe, San Jose, Calif.).

Results

In order to determine whether umbilicus-derived cells could expressepithelial markers, cells were cultured in Chang C medium.Umbilicus-derived cells (P2) were grown in Chang C medium for 11 days.Umbilicus-derived cells stained negative for cytokeratin 18 and keratin8 by immunocytochemistry analysis. Samples grown in Growth Medium werenegative for both markers.

The effect of passage, as well as gelatin and fibronectin substrata wasinvestigated. Cells were grown in Chang C medium for 11 days. RNA andprotein expression of epitheliavhepatocyte-specific proteins wereanalyzed. Immunocytochemistry staining for cytokeratin18, keratin 8,keratin 19, c-met, albumin, desmin, and HNF-1alpha were negative in allconditions. Cells stained positive for vimentin. Expression of bothalbumin and cytochrome p450 2B6 at levels lower than that of human HepG2cells was detected with ASSAYS-ON-DEMAND primers. Albumin and cytochromep450 2B6 expression were also detected in cells grown in Growth Medium.

Umbilicus-derived cells were treated as described in method 1 accordingto a protocol developed by Schwartz et al. (2002) J. Clin. Invest.109(10):1291-1302.). Both albumin and cytochrome p450 2B6 were detectedwith ASSAYS-ON-DEMAND primers at levels lower than HepG2 positivecontrol. No clear pattern emerged between conditions applied and geneexpression levels, i.e., albumin and cytochrome p450 2B6 expression wereeach also detected in control samples. Some expression of albumin andcytochrome p450 2B6 was detected with ASSAYS-ON-DEMAND primers, however,the levels were significantly lower than those observed in HepG2 cells.

Oncostatin M at low concentration of 1 nanogram/milliliter increasedexpression levels of cytochrome p450 2B6 in umbilicus-derived cellsgrown in Growth Medium on gelatin-coated flasks (data not shown). FGF-4and HGF treatment had little effect and may have reduced the expressionof albumin and cytochrome p450 2B6.

Summary. Six protocols were tested for their ability to inducedifferentiation of umbilicus-derived cells to hepatocyte phenotype.Expression of hepatocyte-specific markers, such as albumin andcytochrome p450 2B6 was detected, thereby indicating that the cellsunderwent some differentiation into hepatocytes.

EXAMPLE 19 Adipogenic Differentiation of Umbilicus-Derived Cells

Populations of stem cells have been demonstrated to differentiate intoan adipogenic phenotype (Janderova et al. (2003) Obes. Res. 11(1):65-74;Zangani et al. (1999) Differentiation 64(2):91-101; Liu et al.(2003)Curr. Mol. Med. 3(4):325-40). The potential of umbilicus-derived cellsto differentiate into an adipogenic phenotype was evaluated.

Methods & Materials

Adipose differentiation. Umbilicus-derived cells (P4) were seeded at200,000 cells per well on 6-well tissue culture-treated plates in GrowthMedium. Mesenchymal stem cells (P3, IF2155), osteoblasts (P5, CC2538;Cambrex, Walkerville, Md.), omental cells (P6) (isolated from omentaltissue from NDRI, following protocol used for postpartum-derived cellisolation in Example 1), adipose-derived cells (U.S. Pat. No. 6,555,374B1) (P6), and fibroblasts (P6, CC2509) (Cambrex, Walkerville, Md.) werealso seeded under the same conditions. Prior to initiation ofadipogenesis, Mesenchymal Stem Cells were grown in a Mesenchymal StemCell Growth Medium Bullet kit (Cambrex, Walkerville, Md.). After 2 days,spent medium was aspirated off and cells were washed with phosphatebuffered saline (PBS). Culture medium was then switched to Dulbecco'sMinimal Essential Medium-high glucose (DMEM-Hg; Invitrogen, Carlsbad,Calif.) containing 10 percent FBS (v/v, Hyclone, Logan Utah), 0.02milligrams insulin per milliliter (Sigma, St. Louis, Mo.), and 100 Unitspenicillin per milliliter, 100 milligrams streptomycin per milliliter,0.25 micrograms amphotericin B per milliliter; (Invitrogen, Carlsbad,Calif.). Once the cells had reached confluence, spent medium wasaspirated. Cells were then cultured in an adipose differentiation medium(DMEM-Hg (Invitrogen, Carlsbad, Calif.), containing 10 percent definedfetal bovine serum ((v/v), Hyclone, Logan, Utah), 0.02 milligrams permilliliter insulin (Sigma, St. Louis, Mo.) and 100 units penicillin permilliliter, 100 micrograms streptomycin per milliliter, 0.25 microgramsamphotericin B per milliliter, 5 micromolar isobutylmethylxanthine(Sigma, St. Louis, Mo.), 100 micromolar dexamethasone (Sigma, St. Louis,Mo.), and 2.5 micromolar indomethacin (Sigma, St. Louis, Mo.) for up to4 weeks. Cells were stained with Oil-Red-O to determine the presence oflipid droplet formation.

Red O Staining. Cells were fixed with 10 percent (v/v) neutral bufferedformalin (Richard-Allan Kalamazoo, Mich.). After fixation, the cellswere washed in deionized water and incubated for two minutes inpropylene glycol (absolute; Poly Scientific, Bay Shore, N.Y.). Propyleneglycol was removed by aspiration, and samples were incubated in Oil RedO (Poly Scientific) for one hour. Staining solution was removed byaspiration and stained samples were then incubated in 85 percent (v/v)propylene glycol solution (Poly Scientific) for one minute. Stainedsamples were washed with two changes of de-ionized water. Stainedsamples were counter-stained with Mayer's Hematoxylin (Poly Scientific)and examined with light microscopy. Images were taken at magnificationof 20×.

Leptin Assay. Adipose-derived cells and umbilicus-derived cells wereseeded at 200,000 cells/well in 6-well tissue culture-treated plates.Cells were initially seeded in Growth Medium, which was changed to anadipogenic differentiation medium (DMEM-Hg medium; Invitrogen, Carlsbad,Calif.) containing 1 micromolar dexamethasone (Sigma, St. Louis, Mo.),0.2 millimolar indomethasone (Sigma), 0.01 milligrams per microliterinsulin (Sigma), 0.5 millimolar isobutylmethylxanthine (Sigma), 10percent (v/v) fetal bovine serum (Cat. #SH30070.03; Hyclone, Logan,Utah), 100 Units penicillin per milliliter and 100 microgramsstreptomycin per milliliters (Gibco)). At the end of the assay, theconditioned medium was collected and leptin levels were measured usingan ELISA kit (Quantikine, R&D Systems, Minneapolis, Minn.).

Results

Adipose differentiation. Morphologically MSCs and adipose-derived cellsdemonstrated lipid formation as early as 5 days in this assay. Largeamounts of lipid droplet formation were observed in these cultures by 15days of culture. Cultures of osteoblasts also deposited large amounts oflipid under these conditions after 10 days in culture and extensively at15 days. Lipid droplet formation was observed in umbilicus-derived andomental cell cultures after 15 days of culture. Low level lipid dropletformation was observed in the fibroblast cultures after 20 days inadipogenic-inducing conditions.

Leptin. Leptin was not detected by ELISA in umbilicus-derived cellconditioned medium.

Summary. While leptin was not detected in umbilicus-derived cells byELISA following the adipogenic differentiation protocols used, the dataclearly demonstrate that umbilicus-derived cells undergo a low level ofdifferentiation to an adipocyte phenotype when compared to cultures ofmesenchymal stem cells, adipose-derived cells, or osteoblasts.

EXAMPLE 20 Differentiation into Beta Cell Phenotype

The pancreas contains endocrine cells, organized in islets ofLangerhans, that produce insulin, glucagon, somatostatin, and pancreaticpolypeptide (PP). The ability of umbilicus-derived cells todifferentiate towards cells with an insulin-producing phenotype wastested under eight different induction protocols.

Methods & Materials

Umbilicus-derived cells (various isolates—see below) as well as neonatalor adult Normal Human Dermal Fibroblasts (NHDF) grown in Growth Medium,in gelatin-coated T75 flasks, as well as in different beta-cellpromoting differentiation conditions. Flasks were coated with 2% (w/v)gelatin solution (Sigma, St. Louis, Mo.) for 20 minutes at roomtemperature. Gelatin solution was aspirated off and flasks were washedwith PBS. Basic Fibroblast Growth Factor (bFGF), Epidermal Growth Factor(EGF), Transforming Growth Factor alpha (TGFalpha) and Fibroblast GrowthFactor 10 (FGF-10) were purchased from PeproTech Inc. (Rocky Hill,N.J.). GLP-1 was purchased from Sigma (St. Louis, Mo.)

The following protocols were tested:

Protocol 1:

Cells: Adipose-derived cells (U.S. Pat. No. 6,555,374) andomentum-derived cells, umbilicus-derived cells, (P15), (P17), (P3), andadult Normal Human Dermal Fibroblasts (NHDF) (P10) were utilized. Cellswere maintained under either normal or 5% O₂ conditions. Cells wereseeded at low density (5,000 cells/cm²) in gelatin-coated T75 flasks ongelatin and grown in Ham's F12 medium (Clonetics, Santa Rosa, Calif.),2% (v/v) FBS, penicillin (50 Units/milliliter), streptomycin (50micrograms/milliliter), 10 nanograms/milliliter EGF, 20nanograms/milliliter bFGF until confluence. Confluent cells weretrypsinized and plated at 50,000 cells/cm² in 24-well Tissue CulturePolystyrene (TCPS; BD Biosciences, Bedford, Mass.) plates with orwithout gelatin or collagen coating. Cells were grown in Ham's F12medium, 2% FBS, penicillin (50 Units/milliliter), streptomycin (50micrograms/milliliter), 10 nanograms/milliliter EGF, 20nanograms/milliliter bFGF and 15 nanomolarGLP-1 (7-37 isoform) for up to3 weeks.

Protocol 2:

Cells: Umbilicus-derived cells, isolate 2 (P17), isolate 1 (P15),isolate 4 (P10) and adult NHDF P10 were utilized. Cells were seeded at50,000 cells/cm² 24-well TCP plates and grown in DMEM:Ham's F12 (1:1)medium, B-27 supplement (Gibco, Carlsbad, Calif.), 50 units ofpenicillin per milliliter, 50 milligrams streptomycin per milliliter, 20nanograms/milliliter EGF, 40 nanograms/milliliter bFGF sphericalclusters were generated—usually 4-6 days. Following that period, thespherical clusters were collected, centrifuged, and replated ontolaminin-coated, 24-well plates (BD Biosciences, Bedford, Mass.), andcultured up to 3 weeks in B-27-supplemented medium containing 10nanomolarGLP-1 (7-37) but no other growth factors (i.e. no bFGF and noEGF).

Protocol 3:

Cells: Umbilicus-derived cells, isolate 2 (P17) isolate 1 (P15), isolate4 (P10), and adult NHDF (P10) were utilized. Cells were seeded at highdensity (50,000 cells/cm²) in 24-well TCPS plates and grown inDMEM:Ham's F12 (1:1) medium, B-27 supplement, P/S, 20nanograms/milliliter EGF, 40 nanograms/milliliter bFGF sphericalclusters were generated—usually 4-6 days. Following that period, thespherical clusters were collected, centrifuged, and replated ontolaminin-coated, 24-well plates and cultured up to 3 weeks inB-27-supplemented medium containing 10 nanomolarGLP-1 (1-37 isoform) butno other growth factors (i.e. no bFGF and no EGF).

Protocol 4:

Cells: Adult NHDF (P15), umbilicus-derived, isolate 1 (P18), isolate 2(P21), isolate 3 (P5), isolate 3 (P4), were isolated according to themethod by Mitchell et al. (2). Cells were seeded at 50,000 cells/cm² in24-well TCPS gelatin-coated plates and grown in DMEM:Ham's F12 (1:1)medium, B-27 supplement, penicillin (50 Units/milliliter), streptomycin(50 micrograms/milliliter), 10 nanograms/milliliter FGF-10, and/or 40nanograms/milliliter TGF alpha for more than two weeks.

Protocol 5:

Cells: Adult NHDF, umbilicus-derived, isolate 1 (P18), isolate 2 (P21)isolate 3 (P5), isolate 3 (P4), were isolated according to the method byMitchell et al. (2). Cells were seeded at 50,000 cells/cm² in 24-wellTCPS gelatin-coated plates and grown in EBM-2 medium, 10nanograms/milliliter FGF-10, and/or 40 nanograms/milliliter TGF alphafor greater than two weeks.

Protocol 6:

Cells: Umbilicus-derived, isolate 3 (P2) were utilized. Cells wereseeded at 5,000 cells/cm² in T75 flasks on gelatin and grown either inGrowth Medium or in Ham's F12 medium, 2% FBS, penicillin (50Units/milliliter), streptomycin (50 micrograms/milliliter), 10nanograms/milliliter EGF, 20 nanograms/milliliter bFGF until confluence.Confluent cells were trypsinized and plated at 50,000 cells/cm² in24-well TCPS plates, with or without gelatin coating. Three types ofbasic media were used for up to 3 weeks:

Beta I medium: Ham's F12 medium, 2% FBS, 10 millimolar nicotinamide,penicillin (50 Units/milliliter), streptomycin (50micrograms/milliliter), 25 millimolar glucose;

Beta II medium: Equal parts of DMEM/Ham's F12 media, 2% FBS, 10millimolar nicotinamide, 25 millimolar glucose; and

Endothelial Cell Basal Medium (EBM), (Clonetics, Santa Rosa, Calif.).

The following growth factors were added to each of the media: 10nanograms/milliliter EGF, 20 nanograms/milliliter bFGF, 10 nanomolarGLP-1 (7-37 isoform).

Total RNA isolation and quantitative RT-PCR. RNA was extracted fromumbilicus-derived cells and fibroblasts grown as described in eachprotocol. Cells were lysed with 350 microliters buffer RLT containingbeta-mercaptoethanol (Sigma St. Louis, Mo.) according to themanufacturer's instructions (RNeasy Mini kit, Qiagen, Valencia, Calif.)and RNA extracted according to the manufacturer's instructions (RNeasyMini kit, Qiagen, Valencia, Calif.) with a 2.7 U/sample DNase treatment(Sigma St. Louis, Mo.). RNA was eluted with 50 microliters DEPC-treatedwater and stored at −80° C. RNA was reversed transcribed using randomhexamers with the TaqMan reverse transcription reagents (AppliedBiosystems, Foster City, Calif.) at 25° C. for 10 minutes, 37° C. for 60minutes and 95° C. for 10 minutes. Samples were stored at −20° C.

Real-time PCR. PCR was performed on cDNA samples using ASSAYS-ON-DEMANDgene expression products. PDX-1 (Hs00426216), pro-insulin (Hs00355773),Ngn-3 (Hs00360700) and Glut-2 (Hs00165775) GAPDH (Applied Biosystems,Foster City, Calif.) and TaqMan Universal PCR master mix according tothe manufacturer's instructions (Applied Biosystems, Foster City,Calif.) using a 7000 sequence detection system with ABI Prism 7000 SDSsoftware (Applied Biosystems, Foster City, Calif.). Thermal cycleconditions were initially 50° C. for 2 minutes and 95° C. for 10 minutesfollowed by 40 cycles of 95° C. for 15 seconds and 60° C. for 1 minutes.In addition, another set of primers designed in-house for PDX-1 andNgn-3 were tested. Table 20-1 contains primer sequences. PCR using theseprimers was performed as described above. Pancreas total RNA (Ambion,Austin, Tex.) was used as control. PCR data were analyzed according tothe AACT method recommended by Applied Biosystems (1). TABLE 20-1 Primername Sequence PDX-1 Forward primer 5′-CTGGATTGGCGTTGTTTGTG-3′ (SEQ IDNO:11) PDX-1 Reverse primer 5′-TCCCAAGGTGGAGTGCTGTAG-3′ (SEQ ID NO:12)PDX-1-TaqMan probe 5′-CTGTTGCGCACATCCCTGCCC-3′ (SEQ ID NO:13) Ngn-3Forward primer 5′-GGCAGTCTGGCTTTCTCAGATT-3′ (SEQ ID NO:14) Ngn-3 Reverseprimer 5′-CCCTCTCCCTTACCCTTAGCA-3′ (SEQ ID NO:15) Ngn-3 TaqMan probe5′-CTGTGAAAGGACCTGTCTGTCGC-3′ (SEQ ID NO:16)

Immunofluorescence. Adult human pancreatic tissue was harvested andimmersion fixed in 4% (w/v) paraformaldehyde (Sigma, St. Louis, Mo.)overnight at 4° C. Immunohistochemistry was performed using antibodiesdirected against the following epitopes: insulin (insulin serum; 1:50;LINCO Research, St. Charles, Mo.), PDX-1 (1:50; Santa Cruz Biotech,Santa Cruz, Calif.), glucagon (1:100; Santa Cruz Biotech), somatostatin(1:100; DAKOCytomation, Carpinteria, Calif.), and cytokeratin 18 (CK18;1:400; Sigma, St. Louis, Mo.). Briefly, fixed specimens were blocked offwith a scalpel and placed within OCT embedding compound (Tissue-Tek OCT;Sakura, Torrance, Calif.) on a dry ice bath containing ethanol. Frozenblocks were then sectioned (10 microns thick) using a standard cryostat(Leica Microsystems), and mounted onto glass slides for staining.

Tissue sections were washed with phosphate-buffered saline (PBS) andexposed to a protein blocking solution containing PBS, 4% (v/v) goatserum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100,Sigma) for 1 hour. Primary antibodies, diluted in blocking solution,were then applied to the samples for a period of 4 hours at roomtemperature. Primary antibodies solutions were removed and sampleswashed with PBS prior to application of secondary antibody solutions for1 hour at room temperature containing blocking solution along with goatanti-mouse IgG-Texas Red (1:250; Molecular Probes, Eugene, Oreg.) forCK18, goat anti-rabbit IgG-Alexa 488 (1:250; Molecular Probes) forglucagon and somatostatin, goat anti-guinea pig IgG-FITC (1:150; SantaCruz Biotech) for insulin, or donkey anti-goat IgG-FITC (1:150; SantaCruz Biotech) for PDX-1 staining. Samples were then washed and 10micromolar DAPI (Molecular Probes) applied for 10 minutes to visualizecell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). Representative images werecaptured using a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Results

For umbilicus-derived cells treated according to protocols 1-6,expression of pancreas-specific marker was not detected using real-timePCR and the ASSAYS-ON-DEMAND primers, with the exception that low levelsof Ngn-3 were detected in cells from protocol 6. The same primersproduced positive results with cDNA derived from pancreatic tissue RNA.

Real-time PCR for ngn-3 was performed on cDNA samples derived from humanumbilical cord grown according to protocol 6. PCR was also performedusing ASSAYS-ON-DEMAND Ngn-3 primers (Hs00360700). Humanpancreas-derived cDNA was used as control. No other pancreas-specificmarkers (PDX-1, pro-insulin or Glut-2) were detected with theASSAYS-ON-DEMAND primers.

Experimental conditions in Protocols 2 and 6 applied toumbilicus-derived tissues, but not fibroblasts, produced structuresresembling the cellular assembly of pancreatic epithelial cells intoislets. These structures emerged 3-5 days after the implementation ofthe protocol. However unlike islets, these structures were negative forpancreatic markers (PDX-1, Ngn3, Glut-2 and pro-insulin) expression(tested by real-time PCR).

Pancreas-specific markers were detected in tissue derived from a humanpancreas using immunofluorescence technique and an array of antibodies(see materials and methods). The expression of pancreas-specific markers(e.g. insulin, PDX-1, glucagon, somatostatin, and cytokeratin 18) inhuman pancreatic tissue was readily detectable.

Summary. Limited expression of PDX-1 and Ngn-3 has been observed inumbilicus-derived cells treated with a variety of experimentalprotocols. There were differences in results between in-house designedand commercially available primers. For example, while protocol number 1gave positive data for PDX-1 and Ngn-3 using in-house designed primers,ASSAYS-ON-DEMAND primers for the same genes produced negative data. Theresults were not directly verified by immunological techniques.Notwithstanding such differences, expression of several pancreaticmarkers has been accomplished suggesting the potential ofumbilicus-derived cells to differentiate towards the pancreaticphenotypes.

REFERENCES FOR EXAMPLE 20

-   1. User Bulletin #2 from Applied Biosystems for ABI Prism 7700    Sequence Detection System-   2. Mitchell K E, Weiss M L, Mitchell B M, Martin P, Davis D, Morales    L, Helwig B, Beerenstrauch M, Abou-Easa K, Hildreth T, Troyer D,    Medicetty S. (2003) Matrix cells from Wharton's jelly form neurons    and glia. Stem Cells 21(1):50-60.-   3. H. Edlund. (2002) Pancreatic organogenesis—developmental    mechanisms and implications for therapy. Nat. Rev. Genet. 3:524-532.-   4. S. K. Kim and M. Hebrok. (2001) Intercellular signals regulating    pancreas development and function. Genes Dev. 15:111-127.-   5. Street C N, Rajotte R V, Korbutt G S. (2003) Stem cells: a    promising source of pancreatic islets for transplantation in type 1    diabetes. Curr Top Dev Biol. 58:111-36.

EXAMPLE 21 Chondrogenic Differentiation of Umbilicus-Derived Cells

Cartilage damage and defects lead to approximately 600,000 surgicalprocedures each year in the United States alone (1). A number ofstrategies have been developed to treat these conditions but these havehad limited success. One approach, Cartecel (Genzyme), uses autologouschondrocytes that are collected from a patient, expanded in vitro andthen implanted into the patient (1). This approach has the disadvantageof collecting healthy cartilage and requiring a second procedure toimplant the cultured cells. An alternative possibility is a stemcell-based therapy in which cells are placed at or near the defect siteto directly replace the damaged tissue. Cells may be differentiated intochondrocytes prior to the application or progenitor cells that candifferentiate in situ may be used. Such transplanted cells would replacethe chondrocytes lost in the defect.

Candidate cells for this indication should be evaluated for theirability to differentiate into chondrocytes in vitro. A number ofprotocols have been developed for testing the ability of cells todifferentiate and express chondrocyte marker genes. Umbilicus-derivedcells were tested for their ability to differentiate into chondrocytesin vitro in two different assay systems: the pellet assay culture systemand collagen gel cultures. The pellet culture system has been usedsuccessfully with selected lots of human mesenchymal stem cells (MSC).MSC grown in this assay and treated with transforming growthfactor-beta3 have been shown to differentiate into chondrocytes (2). Thecollagen gel system has been used to culture chondrocytes in vitro (3).Chondrocytes grown under these conditions form a cartilage-likestructure.

Materials and Methods

Cell Culture. Human umbilical cords were received and umbilicus-derivedcells were prepared as described in Example 1 above. Cells were culturedin Growth Medium on gelatin-coated TCP flasks. The cultures wereincubated with 5% CO₂ at 37° C. Cells used in experiments ranged frompassages 4 through 12.

Human articular chondrocytes were purchased from Cambrex (Walkersville,Md.) and cultured in the same medium as the postpartum cells.Twenty-four hours before the experiment, the culture medium was changedto a media containing 1% FBS.

Human mesenchymal stem cells (hMSCs) were purchased from Cambrex(Walkersville, Md.) and cultured in MSCGM (Cambrex). Cells used forexperiments were between passages 2 and 4.

Collagen gel assays. Cultured cells were trypsinized to remove fromculture plate. Cells were washed with centrifugation twice at 300×g for5 minutes in DMEM without serum and counted. Cells were mixed with thefollowing components at the final concentrations listed. Rat tailcollagen (1 milligram/milliliter, BD DiscoveryLabware, Bedford, Mass.),0.01 Normal NaOH and Chondrocyte cell media DMEM, penicillin (100Units/milliliter), streptomycin (100 micrograms/milliliter), 2millimolar L-Glutamine, 1 millimolar Sodium Pyruvate, 0.35 millimolarL-Proline, 100 nanomolardexamethasone, 0.17 millimolar L-Ascorbic Acid,1% (v/v) ITS (insulin, transferring, selenium) (All components fromSigma Chemical Company). The cells were gently mixed with the medium thesamples were aliquoted into individual wells of a 24 well ultra-lowcluster plate (Corning, Corning, N.Y.) at a concentration of either2×10⁵ per well or 5×10⁵ per well. Cultures were placed in an incubatorand left undisturbed for 24 to 48 hours. Medium was replaced with freshchondrocyte media supplemented with appropriate growth factor every 24to 48 hours. Samples were allowed to culture for up to 28 days at whichtime they were removed and fixed in 10% (v/v) formalin (VWR Scientific,West Chester, Pa.) and processed for histological examination. Sampleswere stained with Safranin O or hematoxylin/eosin for evaluation.

Pellet culture assays. Cultured cells were trypsinized to remove fromculture plate. Cells were washed with centrifugation twice at 300×g for5 minutes in DMEM without serum and counted. Cells were resuspended infresh chondrocyte medium (described above) at a concentration of 5×10⁵cells per milliliter. Cells were aliquoted into new polypropylene tubesat 2.5×10⁵ cells per tube. The appropriate samples were then treatedwith TGF-beta3 (10 nanograms/milliliter, Sigma) or GDF-5 (100nanograms/milliliter; R&D Systems, Minneapolis, Minn.). Cells were thencentrifuged at 150×g for 3 minutes. Tubes were then transferred to theincubator at and left undisturbed for 24-48 hours in 5% CO₂ at 37° C.Medium was replaced with fresh chondrocyte cell media and growth factor,where appropriate, every 2-3 days. Samples were allowed to culture forup to 28 days at which time they were removed and fixed and stained asdescribed above.

Results

Pellets were prepared and cultured and described in Methods. Pelletswere grown in medium (Control), or medium supplemented with TGF beta3(10 nanograms/milliliter) or GDF-5 (100 nanograms/milliliter), that wasreplaced every 2 to 3 days. Pellets were collected after 21 days ofculture and stained by Safranin O to test for the presence ofglycosoaminoglycans. The pellets treated with TGFbeta3 and GDF-5 showedsome positive Safranin O staining as compared to control cells. Themorphology of the umbilical cord cells showed some limitedchondrocyte-like morphology.

Summary. Umbilicus-derived cells partially differentiated intochondrocytes in vitro in the pellet culture and the collagen gel assaysystems. The umbilicus-derived cells did show some indications ofglycosaminoglycan expression by the cells. Morphology showed limitedsimilarity to cartilage tissue. These results suggest that conditionscan be optimized to stimulate more complete chondrocyte differentiationof the umbilicus-derived cells.

REFERENCES FOR EXAMPLE 21

-   1. U.S. Markets for Current and Emerging Orthopedic Biomaterials    Products and Technologies. Medtech Insight L.L.C. 2002-   2. Johnstone, B, T. M. Hering, A. I. Caplan, V. M. Goldberg    and J. U. Yoo. In Vitro Chondrogenesis of Bone-Marrow-Derived    Mesenchymal Stem Cells. 1998. Exp Cell Res 238:265-272.-   3. Gosiewska, A., A. Rezania, S. Dhanaraj, M. Vyakarnam, J. Zhou, D.    Burtis, L. Brown, W. Kong, M. Zimmerman and J. Geesin. Development    of a Three-Dimensional Transmigration Assay for Testing Cell-Polymer    Interactions for Tissue Engineering Applications. 2001 Tissue Eng.    7:267-277.

EXAMPLE 22 Further Evaluation of Chondrogenic Potential of Cells Derivedfrom Umbilical Cord Tissue in an In Vitro Pellet Culture Based Assay

Evaluation of the chondrogenic potential of cells derived from umbilicaltissue was performed using in vitro pellet culture based assays. Cellsfrom umbilical cord at early passage (P3) and late passage (P12) wereused. The chondrogenic potential of the cells was assessed in pelletculture assays, under chondrogenic induction conditions in mediumsupplemented with transforming growth factor beta-3 (TGFbeta-3),recombinant human growth and differentiation factor 5 (rhGDF-5), or acombination of both.

Materials & Methods

Reagents. Dulbecco's Modified Essential Media (DMEM), Penicillin andStreptomycin, were obtained from Invitrogen, Carlsbad, Calif. Fetal calfserum (FCS) was obtained from HyClone (Logan, Utah). Mesenchymal stemcell growth medium (MSCGM) and hMSC chondrogenic differentiation bulletkit were obtained from Biowhittaker, Walkersville, Md. TGFbeta-3 wasobtained from Oncogene research products, San Diego, Calif. rhGDF-5 wasobtained from Biopharm, Heidelberg, Germany (WO9601316 A1, U.S. Pat. No.5,994,094 A).

Cells. Human mesenchymal stem cells (Lot# 2F1656) were obtained fromBiowhittaker, Walkersville, Md. and were cultured in MSCGM according tomanufacturers instructions. This lot has been tested previously, and wasshown to be positive in the chondrogenesis assays. Human adult andneonatal fibroblasts were obtained from American Type Culture Collection(ATCC), Manassas, Va. and cultured in Growth Medium on gelatin-coatedtissue culture plastic flasks. Postpartum tissue-derived cells, isolatedfrom human umbilical cords as described in previous examples, wereutilized. Cells were cultured in Growth Medium in a manner similar tothe culture of the fibroblasts. The cell cultures were incubated at 37°C. with 5% CO₂. Cells used for experiments were at passages 3 and 12.

Pellet culture assay. For pellet cultures, 0.25×10⁶ cells were placed ina 15 milliliter conical tube and centrifuged at 150×g for 5 minutes atroom temperature to form a spherical pellet according to protocol forchondrogenic assay from Biowhittaker. Pellets were cultured inchondrogenic induction medium containing TGFbeta-3 (10nanograms/milliliter), rhGDF-5 (500 nanograms/milliliter), or acombination of TGFbeta-3 (10 nanograms/milliliter), and rhGDF-5 (500nanograms/milliliter) for three weeks. Untreated controls were culturedin growth medium. During culture, pellets were re-fed with fresh mediumevery other day. Treatment groups included the following:

Treatment Group

-   -   A. Umbilicus-derived cells early passage (U EP)+rhGDF-5    -   B. Umbilicus-derived cells late passage (U LP)+rhGDF-5, n=2    -   C. Human Mesenchymal Stem cells (HMSC)+rhGDF-5    -   D. Human adult fibroblast cells (HAF)+rhGDF-5    -   E. Umbilicus-derived cells early passage (U EP)+TGFbeta-3    -   F. Umbilicus-derived cells late passage (U LP)+TGFbeta-3, n=2    -   G. Human Mesenchymal Stem cells (HMSC)+TGFbeta-3    -   H. Human adult fibroblast cells (HAF)+TGFbeta-3    -   I. Umbilicus-derived cells early passage (U        EP)+rhGDF-5+TGFbeta-3    -   J. Umbilicus-derived cells late passage (U        LP)+rhGDF-5+TGFbeta-3, n=2    -   K. Human Mesenchymal Stem cells (HMSC)+rhGDF-5+TGFbeta-3    -   L. Human adult fibroblast cells (HAF)+rhGDF-5+TGFbeta-3    -   M. Human neonatal fibroblast cells (HNF)+rhGDF-5+TGFbeta-3    -   N. Umbilicus-derived cells early passage (U EP)    -   O. Umbilicus-derived cells late passage (U LP)    -   P. Human Mesenchymal Stem cells (HMSC)    -   Q. Human adult fibroblast cells (HAF)

Histology of in vitro samples. At the end of the culture period pelletswere fixed in 10% buffered formalin and sent to MPI Research (Mattawan,Mich.) for paraffin embedding, sectioning, and staining with Hematoxylin& Eosin (H&E) and Safranin O (SO) staining.

Results

Umbilicus-derived cells, MSCs and fibroblasts formed cell pellets inchondrogenic induction medium with the different growth factors. Thesize of the pellets at the end of culture period varied among thedifferent cell types. Pellets formed with the umbilical cells tended tobe larger and looser than those formed by MSCs and fibroblasts. Pelletsformed with all cell types and cultured in control medium were smallerthan pellets cultured in chondrogenic induction medium.

Examination of cross sections of pellets stained with H&E and Safranin Oshowed that umbilicus-derived cells at early passage had the potentialto undergo chondrogenic differentiation. Chondrogenesis as assessed bycell condensation, cell morphology and Safranin O positive staining ofmatrix was observed in the umbilical cell pellets cultured inchondrogenic induction medium supplemented with TGFbeta-3, rhGDF-5 orboth. Chondrogenesis in pellets was similar for TGFbeta-3, rhGDF-5 andthe combined treatments. Control pellets cultured in growth mediumshowed no evidence of chondrogenesis. Chondrogenic potential of theumbilicus-derived cells was marginally lower than that observed with theMSCs obtained from Biowhittaker.

Umbilicus-derived cells at late passage did not demonstrate as distincta chondrogenic potential as did early passage umbilical derived cells.However, this may be due to the fact that chondrogenic inductionconditions were optimized for MSCs, not for postpartum derived cells.Some cell condensation was observed with fibroblast, but it was notassociated with Safranin O staining.

EXAMPLE 23 Differentiation to the Cardiomyocyte Phenotype

There is a tremendous need for therapy that will slow the progression ofand/or cure heart disease, such as ischemic heart disease and congestiveheart failure. Cells that can differentiate into cardiomyocytes that canfully integrate into the patient's cardiac muscle without arrhythmiasare highly desirable. Rodent mesenchymal stem cells treated with5-azacytidine have been shown to express markers of cardiomyocytes(Fukuda et al. (2002) C. R. Biol. 325:1027-38). This has not been shownfor adult human stem cells. Additional factors have been used to improvestem cell differentiation including low oxygen (Storch (1990) Biochim.Biophys. Acta 1055:126-9), retinoic acid (Wobus et al. (1997) J. Mol.Cell Cardiol. 29:1525-39), DMSO (Xu et al. (2002) Circ. Res. 91:501-8),and chelerythrine chloride (International PCT Publication No.WO03/025149), which effects the translocation of PKC from the cytosol toplasma membrane and is an inhibitor of PKC activity.

In this example, umbilicus-derived cells were treated with 5-azacytidineeither alone or in combination with DMSO or chelerythrine chloride andmarkers of cardiomyocytes measured by real-time PCR.

Methods & Materials

Cells. Cryopreserved umbilicus-derived cells (P10) were grown in GrowthMedium in gelatin-coated flasks. Cells were seeded at 5×10⁴ cells/wellin 96-well plates in Growth Medium for 24 hours. The medium was changedto 0, 3, 10 and 30 micromolar 5-azacytidine (Sigma, St. Louis, Mo.)alone or with 5 microM chelerythrine chloride (Sigma), 1% (v/v)dimethylsulfoxide (DMSO) (Sigma), or 1 micromolar retinoic acid (Sigma)in MEM-alpha (Sigma), insulin, transferrin, and selenium (ITS; Sigma),10% (v/v) fetal bovine serum, penicillin and streptomycin. The cellswere incubated at 37° C., 5% (v/v) O₂ for 48-72 hours. The medium waschanged to MEM-alpha, insulin, transferrin, and selenium, 10% (v/v)fetal bovine serum, penicillin (50 Units/milliliter) and streptomycin(50 micrograms/milliliter), and cells incubated at 37° C., 5% (v/v) O₂for 14 days.

RNA extraction and Reverse Transcription. Cells were lysed with 150microliters buffer RLT containing beta-mercaptoethanol (Sigma St. Louis,Mo.) according to the manufacturer's instructions (RNeasy 96 kit,Qiagen, Valencia, Calif.) and stored at −80° C. Cell lysates were thawedand RNA extracted according to the manufacturer's instructions (RNeasy96 kit, Qiagen, Valencia, Calif.) with a 2.7 Units/sample DNasetreatment (Sigma St. Louis, Mo.). RNA was eluted with 50 microlitersDEPC-treated water and stored at −80° C. RNA was reverse-transcribedusing random hexamers with the TaqMan reverse transcription reagents(Applied Biosystems, Foster City, Calif.) at 25° C. for 10 minutes, 37°C. for 60 minutes and 95° C. for 10 minutes. Samples were stored at −20°C.

PCR. PCR was performed on cDNA samples using ASSAYS-ON-DEMAND geneexpression products cardiac myosin (Hs00165276 ml), skeletal myosin(Hs00428600), GATA 4 (Hs00171403 ml), GAPDH (Applied Biosystems, FosterCity, Calif.), and TaqMan Universal PCR master mix according to themanufacturer's instructions (Applied Biosystems, Foster City, Calif.)using a 7000 sequence detection system with ABI Prism 7000 SDS software(Applied Biosystems). Thermal cycle conditions were initially 50° C. for2 minutes and 95° C. for 10 minutes followed by 40 cycles of 95° C. for15 seconds and 60° C. for 1 minute. cDNA from heart and skeletal muscle(Ambion, Austin Tex.) were used as controls.

Results

Control RNA from cardiac muscle showed expression of cardiac myosin andGATA 4, skeletal muscle RNA showed skeletal myosin and cardiac myosin,but no GATA 4 expression. Umbilicus-derived cells (P12) treated for 48 hwith factors and cultured for a further 14 days expressed low levels ofGATA 4, but no skeletal myosin or cardiac myosin. Additional samplesfrom umbilicus-derived cells also showed expression of GATA 4.

Summary. Untreated umbilicus-derived cells constitutively express GATA4, a nuclear transcription factor in cardiomyocytes, sertoli cells, andhepatocytes.

EXAMPLE 24 Assessment of Umbilicus-Derived Cells for CardiovascularTherapy in a Rodent Coronary Ligation Model

Animal models of heart failure have facilitated the understanding of thepathophysiology of the disease and have assisted in the development ofnew treatments for congestive heart failure (CHF). Coronary arteryligation, or the blocking of the vessels that supply the heart tissue,in the rat closely mimics the pathophysiology of acute myocardialinfarction in humans and has been used successfully to studypharmacological interventions for CHF. Cell transplantation of humancells into cardiac lesions is a potential viable therapeutic treatmentfor CHF.

The efficacy of intracardiac human umbilicus-derived cell treatment whenadministered 15 minutes post-coronary artery occlusion was evaluated ina rodent model of myocardial ischemia/infarction.

Methods & Materials

The Charles River Worcester, Mass. test facility is accredited by theAssociation for the Assessment and Accreditation of Laboratory AnimalCare, International (AAALAC) and registered with the United StatesDepartment of Agriculture to conduct research in laboratory animals. Allthe conditions of testing conformed to the Animal Welfare Act (9 CFR)and its amendments. The protocol was reviewed and approved by theInstitutional Animal Care and Use Committee (IACUC) at the Test Facilityfor compliance with regulations prior to study initiation.

The animals having characteristics identified in Table 25-1 wereindividually housed in micro-isolator cages on autoclaved bedding. Thecages conformed to standards set forth in The Guide for the Care and Useof Laboratory Animals. TABLE 25-1 Animal characteristics Species: Rattusnorvegicus Strain: Rnu Source: Charles River Laboratories Age at Dosing:6-8 weeks Weight at Dosing: ˜200-250 grams Number of Males (includingspares): 40 + 10

Purina Certified Diet (irradiated) was provided to the animals adlibitum. This diet was routinely analyzed by the manufacturer fornutritional components and environmental contaminants. Results of themanufacturer's analyses are on file at the Test Facility.

Autoclaved filtered tap water was provided ad libitum. Samples of thefiltered water were analyzed for total dissolved solids, hardness,specified microbiological content, and selected environmentalcontaminants. Results of these analyses are on file at the TestFacility.

Environmental controls were set to maintain temperatures of 18 to 26° C.(64 to 79° F.) with relative humidity at 30% to 70%. A 12:12 hourlight:dark cycle was maintained. Ten or greater air changes per hourwere maintained in the animal rooms. Upon receipt and prior to use onthe study, the animals were held for a minimum of four days forconditioning according to the Test Facility Vendor Management Program asdescribed in the Test Facility Standard Operating Procedure, Receipt,Conditioning, and Quarantine of Laboratory Animals.

Each animal was identified by a unique number indicated by an ear punch.Animals were randomly assigned to groups by a weight-ordereddistribution such that individual body weights did not exceed ±20% ofmean weight.

The animals were anesthetized with sodium pentobarbital (40milligrams/kilogram) and buprenorphine(0.05 milligrams/kilogram) as asingle cocktail given intramuscularly (IM). Following the establishmentof anesthesia, animals were intubated using 18 to 16 gauge, 2-inchlength angiocaths, or appropriate sized angiocath, and maintained onroom air respiration (supplemented with oxygen) and a positive pressureventilator throughout the surgical procedure. Additional anesthesia wasgiven incrementally as needed. Preoperative antibiotic therapy was alsoadministered, Benzathine/Procaine penicillin G, 40,000 Units/kilogram,IM. Additional antibiotic therapy was administered every 48 hours.

Electrode pads were placed around the appropriate paws of the animals toreceive a useable ECG signal. Animals were positioned on a heating padto help maintain body temperature throughout the procedure. A rectaltemperature probe was inserted into the animal to monitor bodytemperature. Ophthalmic ointment was administered to each eye. Thesurgical sites (thoracic area) were prepared for aseptic surgery byremoving any excess fur, and gently wiping the area with sponges soakedin 70% isopropyl alcohol, which was allowed to dry. Iodone (MEDISEPPS,or similar solution) was then applied to the area and allowed to dry.The area was appropriately draped for strict aseptic surgery.

A surgical incision was made on the skin over the fourth intercostalspace. Blunt dissection through the muscle layers was used to access thethoracic cavity. A retractor was carefully inserted into the fourthintercostal space and opened to allow access to the interior cavity. Thepericardium was carefully opened via gentle teasing with cotton swabsdampened in sterile saline solution. A damp cotton swab was used togently push the apex of the heart into the opening where a length of 6-0silk suture was attached into the myocardium for manipulation of theheart. After a pause to allow the heart to recover, the suture placed inthe apex was used to ease the heart out of the chest cavity and to placesufficient tension on the heart to allow access to the upper heart andthe left anterior descending coronary artery (LAD). Another length of6-0 silk suture was placed into the myocardium so as to surround theLAD. The pressure on the apical suture was released and the heartallowed to return to the interior of the chest cavity.

Once the heart rate and ECG returned to baseline values, the ligaturesaround the LAD were tied off to occlude the LAD. This was a permanentocclusion with the suture tied off and the ends trimmed. After theligature was tied, the surgeon looked for the following indications ofsuccessful occlusion: change in color of the area of the heart directlybelow the ligature to a white/grayish white as a result of thetermination of blood flow to the area and a significant change in theECG corresponding to occlusion of the LAD. Arrhythmias may havedeveloped within the first 10 minutes of the occlusion. The rat wasmonitored closely during this time period in the event thatresuscitation was necessary. In the event of severe arrhythmia andfailure of the rat to convert to normal sinus rhythm without assistance,aid was rendered via cardiac massage. Approximately 15 minutes followingthe initiation of the LAD occlusion, the area of left ventricle madeischemic was treated with either vehicle or test article by directinjection into the ischemic myocardium. Treatment consisted of three toten intramyocardial injections (100 uL/injection) into the ischemic zoneof myocardium.

Human cells were grown in Growth Medium in gelatin-coated T300 flasks.Cells were washed with phosphate buffered saline (PBS, Gibco, CarlsbadCalif.) and trypsinized using Trypsin/EDTA (Gibco, Carlsbad Calif.). Thetrypsinization was stopped by adding Growth Medium. The cells werecentrifuged at 150×g, supernatant removed, and the cell pellet wasresuspended in approximately 1 milliliter Growth Medium per millioncells. An aliquot of cells was removed and added to trypan blue (Sigma,St. Louis, Mo.). The viable cell number was estimated using ahemocytometer. The cell suspension was centrifuged and resuspended in 1milliliter Growth Medium containing 10% (v/v) DMSO (Hybrimax, Sigma, St.Louis, Mo.) per 5 million cells and transferred into Cryovials(Nalgene). The cells were cooled at approximately 1° C./minute overnightin a −80° C. freezer using a “Mr. Frosty” freezing container (Nalgene,Rochester, N.Y.). Vials of cells were transferred into liquid nitrogen.Vials were shipped from CBAT, Somerville, N.J. to Charles River,Worcester, Mass. on dry ice and stored at −80° C. Approximately 1-2hours before injection of cells into the animal, a vial of cells wasthawed rapidly in a 37° C. water bath. Under aseptic conditions in aBSL2 biosafety cabinet, cells were added to 40 milliliters PBS withmagnesium and calcium (Sigma St. Louis, Mo.) and centrifuged at 150×gfor 5 minutes before resuspending the cell pellet in 10 milliliters PBS.The cell number and viability was estimated as described above. Thecells were centrifuged at 150×g for 5 minutes and resuspended in PBS ata final concentration of 10⁶ viable cells/100 microliters. The cellsuspension was loaded into 1 milliliter syringes with a 30G needle andkept on ice. Viability was assessed again up to 5 hours on ice.

Following the administration of treatment (Table 24-1) and stabilizationof the heart, the surgeon began closing the surgical incision. Theretractor was removed. The lungs were over-inflated for 3 to 4 breathsand visually inspected as much as possible to ensure that they werefully re-inflated. This created a negative pressure necessary to preventpneumothorax post-recovery. To evacuate fluid and excess air from thethoracic cavity after closing the cavity, an intravenous catheter (i.e.,20 gauge, 2 millimeters in length) was placed through the skin andmuscle layers so that the tip remains in the thoracic cavity. Care wastaken so that the tip did not pierce the lung or heart. The separatedribs and associated muscle was sutured together with appropriate suture.The upper layers of muscle was sutured using a simple continuouspattern. The skin was closed with 4-0 silk using a horizontal mattresspattern. A 10 milliliter syringe was attached to the intravenouscatheter that had been previously placed in the thoracic cavity and theplunger slowly pulled back to withdraw fluids and air from the cavity.At the same time, the catheter was slowly withdrawn from the entry site,thereby allowing the surrounding muscle mass and skin to seal thepuncture. The surgical drape was removed and fluids (i.e., lactatedRingers solution, 25 milliliters/kilogram subcutaneously [SC] orintraperitoneally [IP]) were given. TABLE 24-1 Treatment regimens DosageTime of Gr. No. of Level Dose Conc. Route/Dose Treatment Necropsy No.Males Test Article (cells/animal) (cells/mL) Regimen Administration Day1 8 Vehicle 0 0 Direct 15 minutes Day 28 2 8 Umbilical (P10) 1 million10 million injection(s) into after coronary (±1 Day) (B) the ischemicartery ligation 2 8 Human region of the left fibroblasts ventricle ofthe 1F1853 (P10) (D) heart, consisting of 3 to 10 intramyocardialinjections of 100 μL total.Gr. = Group;No. = Number;Conc. = Concentration

Immediately after each rat had undergone treatment with test article andthe incision was sutured, the animal underwent an echocardiography (ECG)examination. Anesthesia was maintained throughout the completion of theecho examination. Upon the completion of the echo examination,ventilation was discontinued, and the rat was returned to the recoveryarea to recover in a heated, oxygenated recovery cage.

A second echo examination of each surviving animal was completed at theend of the study (approximately 28 days post-treatment), prior totermination. During the second examination, the animals wereanesthetized as described previously.

For each echo examination, the left thoracic area was shaved, andwarmed, ultrasonic gel was applied to the skin to enhance contact withthe transducer. Electrode pads were placed around the appropriateextremities to receive an ECG signal. Echocardiographic images includedshort axis and long axis views to allow for the determination ofventricular cavity dimensions, contractility, blood flow throughvasculature, and wall thickness. These images were saved on optical diskfor further analysis. After examination, the gel medium was removed fromthe skin with gauze or paper towel. The rat was removed from theventilator and placed in a warmed recovery cage until mobile.

At the conclusion of the surgical procedures, respiratory ventilationwas turned off. The animals were observed for pedal reflex. The rectalprobe and ECG electrodes subsequently were removed, and the animal wasextubated and placed in a warmed oxygenated recovery cage. Aftercomplete recovery from anesthesia, the animals were given buprenorphine(0.05 milligram/kilogram, SC). Observations were made regularly untilthe animals showed full mobility and an interest in food and water. Theanimals then were placed in a clean housing cage and returned to theanimal housing room. Animals were monitored for surgical incisionintegrity twice daily post-surgery.

Analgesics (i.e., Buprenorphine, 0.05 milligram/kilogram SC.) were giventwice daily for 4 days post-operatively and thereafter as needed. Visualindications of post-operative pain include lack of normal body posturesand movement (e.g., animal remains in hunched position), antipathy, lackof eating/drinking, lack of grooming, and the like.

Body weight was recorded for each animal prior to initial treatment,weekly thereafter, and on the day of necropsy. Animals found dead wereweighed and necropsied.

In order for the heart to be harvested, each rat was anesthetized as wasdone for surgery. The jugular vein was cannulated. The heart wasarrested in diastole with KCl infused via the jugular cannula. The heartwas then removed from the thoracic cavity. A limited necropsy was thenperformed on the heart after which the heart was placed in 10% neutralbuffered formalin. The remainder of each carcass was then discarded withno further evaluation.

Hearts of all animals that were found dead or euthanized moribund wereplaced in 4% paraformaldehyde until evaluated. The remainder of eachcarcass was then discarded with no further evaluation.

Histology and Image Analysis. Fixed tissues sectioned with a stainlesssteel coronal heart matrix (Harvard Apparatus, Holliston, Mass.) yieldedfour two-millimeter thick serial tissue sections. Sections wereprocessed and serially embedded in paraffin using routine methods.Five-micron sections were obtained by microtome and stained withMasson's Tri-Chrome for Connective Tissue (Poly Scientific, Bay Shore,N.Y.) using the manufacturer's procedures. Electronic photomicrographswere captured and analyzed using image analysis methods developed byPhase 3 Imaging System (Glen Mills, Pa.). Photomicrographs of thetri-chrome stained sections were calorimetrically analyzedelectronically to determine the overall area of the ventricle and freewall and the area of the differential staining.

Results

There was no loss in the viability of cells over 5 hours in the vehiclewhen kept on ice. Cells were injected into the infarct with one to threeneedle entry points and multiple changes in direction of needleorientation.

Fractional shortening values were calculated as described by Sahn et al.(1978) Circulation 58:1072-1083. The fractional shortening of thevehicle-treated animals had a significant decrease from 47.7%±8.3% atDay 0 to 23.5%±30.2% at Day 28 (p<0.05). The animals that were treatedwith umbilicus-derived cells showed small, non-significant differencesbetween the fractional shortening between Day 0 and 28. There were nosignificant differences between the fractional shortening between thetreatment groups at Day 0.

Upon termination of the study, hearts were collected and subjected tohistological analysis. The hearts were arrested in diastole and fixed.The results were calculated from an algorithm to estimate the percentageof total heart area that comprises the infarct. The infarct size in thevehicle-treated animals was 22.9%±6.7% of heart area, while the infarctsize in hearts treated with with umbilical cord cells was 12.5%±2.5%,with placenta-derived cells (isolate 2) was 12.9%±3.4%, and withfibroblasts was 19.3%±8.0%. The difference of infarct size ofcell-treated animals relative to vehicle-treated animals was notstatistically significant based on Student's t-test.

Summary. The results of the present study suggest that theumbilicus-derived cells have some benefit in reducing the damage of asurgically induced myocardial infarction in rats. The vehicle-treatedanimals showed a significant reduction in cardiac function from day 0 today 28, as measured by fractional shortening, while theumbilicus-derived cell-treated animals showed minimal change over the28-day study. The fibroblast-treated animals showed minimal change butonly two animals survived the study. Evaluation of infarct sizesuggested that there is some modest, but not statistically significant,reduction in the infarct size in the postpartum-derived cell-treatedanimals as compared to the vehicle controls at Day 28. Taken together,these data support efficacy of the umbilicus-derived cells in reducingdamage from a myocardial infarction.

EXAMPLE 25 Use of Umbilicus-Derived Cells in the Treatment of RetinitisPigmentosa

Currently no real treatment exists for blinding disorders that stem fromthe degeneration of cells in the retina. Loss of photoreceptors as aresult of apoptosis or secondary degeneration lead to progressivedeterioration of vision, and ultimately to blindness. Diseases in whichthis occurs include age-related macular degeneration (AMD) and retinitispigmentosa (RP). RP is most commonly associated with a single genemutation, which contributes to photoreceptor cell death.

The retinal photoreceptors and adjacent retinal pigment epithelium forma functional unit. The Royal College of Surgeons (RCS) rat presents witha tyrosine receptor kinase (Merkt) defect affecting outer segmentphagocytosis, leading to photoreceptor cell death (1). Transplantationof retinal pigment epithelial (RPE) cells into the subretinal space ofRCS rats was found to limit the progress of photoreceptor loss andpreserve visual function (2). In this example, it is demonstrated thatumbilicus-derived cells can be used to promote photoreceptor rescue inan RCS model.

Methods & Materials

Cell transplants. Cultures of human adult umbilical and fibroblast cells(passage 10) were expanded for 1 passage. All cells were initiallyseeded at 5,000 cells/cm² on gelatin-coated T75 flasks in Growth Medium.For subsequent passages, all cells were treated as follows: Aftertrypsinization, viable cells were counted after trypan blue staining.Briefly, 50 microliters of cell suspension was combined with 50microliters of 0.04% w/v trypan blue (Sigma, St. Louis Mo.) and theviable cell number, was estimated using a hemocytometer. Cells weretrypsinized and washed three times in supplement-free DMEM:Low glucosemedium (Invitrogen, Carlsbad, Calif.). Cultures of human umbilical andfibroblast cells at passage 11 were trypsinized and washed twice inLeibovitz's L-15 medium (Invitrogen, Carlsbad, Calif.).

For the transplantation procedure, dystrophic RCS rats were anesthetizedwith xylazine-ketamine (1 milligram/kilogram i.p. of the followingmixture: 2.5 milliliters xylazine at 20 milligrams/milliliter, 5milliliters ketamine at 100 milligrams/milliliter, and 0.5 milliliterdistilled water) and their heads secured by a nose bar. Cells devoid ofserum were resuspended (2×10⁵ cells per injection) in 2 microliters ofLeibovitz, L-15 medium (Invitrogen, Carlsbad, Calif.) and transplantedusing a fine glass pipette (internal diameter 75-150 microns)trans-scerally.

Cells were delivered into the dorso-temporal subretinal space ofanesthetized 3 week old dystrophic-pigmented RCS rats (total N=10/celltype). Cells were injected unilaterally into the right eye, while theleft eye was injected with carrier medium alone (Sham control;Leibovitz's L-15 medium). Viability of residual transplant cellsremained at greater than 95% as assessed by trypan blue exclusion at theend of the transplant session. After cell injections were performed,animals were injected with dexamethasone (2 milligram/kilogram) for 10days post transplantation. For the duration of the study, animals weremaintained on oral cyclosporine A (210 milligrams/liter of drinkingwater; resulting blood concentration: 250-300 micrograms/liter) (BedfordLabs, Bedford, Ohio) from 2 days pre-transplantation until end of thestudy. Food and water were available ad libitum. Animals were sacrificedat 60 or 90 days postoperatively, with some animals being euthanitizedat earlier timepoints for histological assessment of short-term changesassociated with cell transplantation.

ERG recordings. Following overnight dark adaptation, animals wereprepared for ERG recording under dim red light, as previously described(3). In brief, under anesthesia (with a mixture of 150milligram/kilogram i.p ketamine, and 10 milligram/kilogram i.p.xylazine) the animal's head was secured with a stereotaxic head holderand the body temperature monitored through a rectal thermometer andmaintained at 38° C. using a homeothermic blanket. Pupils were dilatedusing equal parts of topical 2.5% phenylephrine and 1% tropicamide.Topical anesthesia with 0.75% bupivacaine was used to prevent anycorneal reflexes and a drop of 0.9% saline was frequently applied on thecornea to prevent its dehydration and allow electrical contact with therecording electrode (gold wire loop). A 25-gauge needle inserted underthe scalp, between the two eyes, served as the reference electrode.Amplification (at 1-1,000 Hz bandpass, without notch filtering),stimulus presentation, and data acquisition were provided by theUTAS-3000 system from LKC Technologies (Gaithersburg, Md.). ERGs wererecorded at 60 and 90 days of age in the umbilical cell groups and at 60days only in the fibroblast groups.

Mixed a- and b-wave recording. For the quantification of dark-adaptedb-waves, recordings consisted of single flash presentations (10microseconds duration), repeated 3 to 5 times to verify the responsereliability and improve the signal-to-noise ratio, if required. Stimuliwere presented at six increasing intensities in one log unit stepsvarying from −3.6 to 1.4 log candila/m² in luminance. To minimize thepotential bleaching of rods, inter-stimulus intervals were increased asthe stimulus luminance was elevated from 10 seconds at lowest stimulusintensity to 2 minutes at highest stimulus intensity. The maximum b-waveamplitude was defined as that obtained from the flash intensity series,regardless of the stimulus intensity. The true V_(max) from fitting thedata with a Naka-Rushton curve was not used because ERG responses wereoften erratic at higher luminance levels in dystrophic animals andshowed tendencies for depressed responses around 0.4 and 1.4 logcandila/m². In order to determine the age at which ERG components wereobtained or lost, criterion amplitudes were used: 20 microVolts for a-and b-waves, and 10 microVolts for STR-like responses. The amplitude ofthe b-wave was measured from the a-wave negative peak up to the b-wavepositive apex, and not up to the peak of oscillations, which can exceedthe b-wave apex (4).

Isolation of rod and cone responses. The double flash protocol was usedto determine the isolation of rod and cone responses (5). A probe flashwas presented 1 second after a conditioning flash, using a specificfeature of the UTAS-3000 system (LKC Technologies) with calibratedganzfeld; assuring complete recharge of the stimulator under theconditions used. The role of the conditioning flash in the procedure wasto transiently saturate rods so that they were rendered unresponsive tothe probe flash. Response to the probe flash was taken as reflectingcone-driven activity. A rod-driven b-wave was obtained by subtractingthe cone-driven response from the mixed response (obtained followingpresentation of a probe flash alone, i.e. not preceded by anyconditioning flash).

Functional Assessment. Physiological retinal sensitivity testing wasperformed to demonstrate retinal response to dim light. Animals wereanesthetized with a recovery dose of urethane at 1.25 grams/kilogrami.p. Physiological assessment in the animals was tested post graft inanimals at 90 days by recording multiunit extracellular activity in thesuperior colliculus to illumination of respective visual receptivefields (6). This procedure was repeated for 20 independent points(spaced 200 millimeters apart, with each step corresponding toapproximately 10-150 displacements in the visual field), covering thevisual field. Visual thresholds were measured as the increase inintensity over background and maintained at 0.02 candila/m²(luminescence unit) [at least 2.6 logarithm units below rod saturation(7)], required for activating units in the superficial 200 microns ofthe superior colliculus with a spot of light 3° in diameter. Responseparameters were compared between transplanted and sham control eyes thatreceived vehicle alone.

Histology. Animals were sacrificed with an overdose of urethane (12.5grams/kilogram). The orientation of the eye was maintained by placing a6.0 suture through the superior rectus muscle prior to enucleation.After making a corneal incision, the eyes were fixed with 2.5%parafomaldehyde, 2.5% glutaraldehyde, 0.01% picric acid in 0.1 Mcacodylate buffer (pH 7.4). After fixation, the cornea and lens wereremoved by cutting around the cilliary body. A small nick was made inthe periphery of the dorsal retina prior to removal of the superiorrectus to assist in maintaining orientation. The retinas were thenpost-fixed in 1% osmium tetroxide for 1 hour. After dehydration througha series of alcohols to epoxypropane, the retinas were embedded in TAABembedding resin (TAAB Laboratories, Aldemarston, UK). Semi-thin sectionswere stained with 1% Toluidine Blue in 1% borate buffer and the ultrathin sections were contrasted with uranyl acetate and lead citrate.

For Nissl staining, sections were stained with 0.75% cresyl violet(Sigma, St. Louis, Mo.) after which they were dehydrated through gradedalcohols at 70, 95 and 100% twice, placed in xylene (Sigma, St. Louis,Mo.), rinsed with PBS (pH 7.4) (Invitrogen, Carlsbad, Calif.),coverslipped and mounted with DPX mountant (Sigma, St. Louis, Mo.).

Results

ERG Recordings. Animals that received umbilicus-derived cell injectionsexhibited relative preservation of visual response properties 60 and 90days post-operatively (Table 25-1). The response observed in theseanimals was greater than that seen with fibroblast or sham treatedanimals.

Umbilicus-derived cell-transplanted animals (n=6) demonstrated goodimprovement in all outcome measures tested at 60 days (Table 25-1),a-wave (27±11) versus sham controls (0), mixed b-wave (117±67) versussham controls (18±13), cone-b-wave (55±25) versus sham controls (28±11),and in rod contribution (49±16%) versus sham controls (6±7%).Furthermore, at 90 days, improved responses were measured in two animalstested, with measures including: a-wave (15±7) versus sham controls (0),mixed b-wave (37±15) versus sham controls (0), cone-b-wave (16±11)versus sham controls (7±5), and in rod contribution (58±39%) versus shamcontrols (0%). These results indicate that visual responsiveness wasimproved in umbilicus-derived cell transplanted animals with evidencefor photoreceptor rescue. Although a diminution in responsiveness to ERGwas observed in the 90-day animals tested, their preservation of visualfunction in comparison to sham-treated controls was good.

In contrast to umbilicus-derived cells, fibroblast transplantationsshowed no improvement in any of the parameters tested. TABLE 25-1 ERGdata a-wave mixed b-wave cone b-wave % rod contribution Group UntreatedTreated Untreated Treated Untreated Treated Untreated Treated Sham 60 d0 0 7 ± 9 0 23 ± 5  12 ± 16 N/A N/A U (n = 6) 0 27 ± 11 18 ± 13 117 ±67  28 ± 11 55 ± 25 6 ± 7 49 ± 16 60 d U (n = 6) 0 15 ± 7  0 37 ± 15 7 ±5 16 ± 11 0 58 ± 39 90 dN.B.Sham = control (medium only),U = Umbilicus-derived cell transplant

Histology. Following transplantation, there was no histological evidenceof an inflammatory reaction and infiltrating immune cells were notobserved in Nissl-stained sections in the postpartum cell groups.However, fibroblast implantations resulted in animal death (n=7) andindications of early stage inflammatory responses. Histologically at the90 day time point in the umbilicus-derived cell transplanted animalsanatomical rescue of photoreceptors was clearly demonstrated. Thephotoreceptors formed a thick layer separated by a gap from the innernuclear layer, made up of other retinal cells. By comparison, the widthof the outer layer in the sham control was, at best, a discontinuoussingle layer as opposed to around 5 cells thick in the grafted eye. Incomparison to a normal animal this is marginally more than half thethickness of photoreceptor cell layers normally observed.

Functional Assessment. Efficacy of transplants in preventing visual losswas monitored by assessment of electrophysiological responsiveness intwo animals. The threshold sensitivity response to light was used todefine the area of visual field rescue in sham-injected control eyesversus eyes transplanted with umbilicus-derived cells. In nondystrophicrats, visual thresholds never exceeded 0.5 log candila/m² abovebackground. In non-operated dystrophic rats, the thresholds are usuallyin the magnitude of 4 log candila/m² units (8). By contrast, innon-operated sham injected dystrophic rats, the thresholds were in theorder of 2.9-4.9 log candila/m² units with an average threshold of 4.0log candila/m² units, in some instances no recording could be attained.Thus, the sham-injected rats showed some highly localized functionalrescue in the temporal retina. However, the human umbilicus-derived celltransplanted rats exhibited substantially greater levels of visualpreservation with thresholds ranging from 0.8 to 2.1 log candila/m²units, with an average threshold of 1.3 log candila/m² units.

Summary. Transplantation of umbilicus-derived cells into dystrophic RCSrats can preserve photoreceptors. In this degenerative model, one wouldexpect the a-wave to disappear within 30 to 60 days and the b-wave todisappear within 3 months. Thus, the basically retained a-wave indicatesthat real and normal rod function is preserved. Rod contribution tob-wave suggests abnormal rod function is still possible. The sustainednon-rod b-wave is the measure of how much cone function is maintained,which is a real measure of vision. Thus, the level of improvementassessed both physiologically and anatomically followingumbilicus-derived cell transplantation is well defined here. ERGmeasurements provide an assessment of visual function afterphotoreceptor loss, indicating changes in electrical activity in theretina. However, ERG does not provide direct information as to imageforming capability. The measurement of collicular threshold sensitivityused in this study provides an indication of relative preservation ofvisual fields. The importance of this measure is based on a correlationbetween the amounts of functional rescue and anatomical preservation andthat the data collected compares with visual field perimetry testing inhumans (9). The transplantation has demonstrated a retardation of thedisease process in the test animals. Thus, the results presented hereindemonstrate clear evidence of functional efficacy of grafting humanumbilicus-derived cells into the subretinal space, and that preservationof photoreceptors occurs in the general region in which the graftedcells are located.

REFERENCES FOR EXAMPLE 25

-   1. D'Cruz P M, Yasumura D, Weir J, Matthes M T, Abderrahim H, LaVail    M M, Vollrath D. Mutation of the receptor tyrosine kinase gene Mertk    in the retinal dystrophic RCS rat. Hum Mol Genet. 2000 Mar.    1;9(4):645-51.-   2. Li L X, Turner J E. Inherited retinal dystrophy in the RCS rat:    prevention of photoreceptor degeneration by pigment epithelial cell    transplantation. Exp Eye Res. 1988 December;47(6):911-7.-   3. Sauve, Y, Lu, B and Lund R D. The relationship between full field    electroretinogram and perimetry-like visual thresholds in RCS rats    during photoreceptor degeneration and rescue by cell transplants.    Vision Res. 2004 January;44(1):9-18.-   4. Nusinowitz, S., Ridder, W H 3rd, and Heckonlively, H R. Rod    multifocal electroretinograms in mice. Invest Ophthalmol Vis Sci.    1999 November;40(12):2848-58.-   5. Nixon, P J, Bui, P V, Armitage, J A, and Vingrys A J. The    contribution of cone responses to rat electroretinograms. Clin    Experiment Ophthalmol. 2001 June;29(3):193-6.-   6. Lund R D, Adamson P, Sauve Y, Keegan D J, Girman S V, Wang S,    Winton H, Kanuga N, Kwan A S, Beauchene L, Zerbib A, Hetherington L,    Couraud P O, Coffey P, Greenwood J. Subretinal transplantation of    genetically modified human cell lines attenuates loss of visual    function in dystrophic rats. Proc Natl Acad Sci USA. 2001 Aug.    14;98(17):9942-7.-   7. Siminoff R, Kruger L. Properties of reptilian cutaneous    mechanoreceptors. Exp Neurol. 1968 March;20(3):403-14.-   8. Blakema, G. W. and Drager, U. C. 1991. Visual Neuroscience.    6:577-585.-   9. Beck R W, Bergstrom T J, Lichter P R. A clinical comparison of    visual field testing with a new automated perimeter, the Humphrey    Field Analyzer, and the Goldmann perimeter. Ophthalmology. 1985    January;92(1):77-82.

EXAMPLE 26 Chondrogenic Potential of Postpartum-Derived Cells onImplantation in SCID mice

The chondrogenic potential of cells derived from umbilicus or placentatissue was evaluated following seeding on bioresorbable growthfactor-loaded scaffolds and implantation into SCID mice.

Materials & Methods

Reagents. Dulbecco's Modified Essential Media (DMEM), Penicillin andStreptomycin, were obtained from Invitrogen, Carlsbad, Calif. Fetal calfserum (FCS) was obtained from HyClone (Logan, Utah). Mesenchymal stemcell growth medium (MSCGM) was obtained from Biowhittaker, Walkersville,Md. TGFbeta-3 was obtained from Oncogene research products, San Diego,Calif. rhGDF-5 was obtained from Biopharm, Heidelberg, Germany(International PCT Publication No. WO96/01316 A1, U.S. Pat. No.5,994,094A). Chondrocyte growth medium comprised DMEM-High glucosesupplemented with 10% fetal calf serum (FCS), 10 millimolar HEPES, 0.1millimolar nonessential amino acids, 20 micrograms/milliliter L-proline,50 micrograms/milliliter ascorbic acid, 100 Units/milliliter penicillin,100 micrograms/milliliter streptomycin, and 0.25 micrograms/milliliteramphotericin B. Bovine fibrinogen was obtained from Calbiochem.

Cells. Human mesenchymal stem cells (hMSC, Lot# 2F1656) were obtainedfrom Biowhittaker, Walkersville, Md. and were cultured in MSCGMaccording to the manufacturer's instructions. This lot was tested in thelaboratory previously in in vitro experiments and was shown to bepositive in the chondrogenesis assays. Human adult fibroblasts wereobtained from American Type Culture Collection (ATCC, Manassas, Va.) andcultured in Growth Medium on gelatin-coated tissue culture plasticflasks. Postpartum-derived cells isolated from human umbilical cords(Lot# 022703Umb) and placenta (Lot# 071003Plac) were prepared aspreviously described (Example 1). Cells were cultured in Growth Media ongelatin-coated tissue culture plastic flasks. The cell cultures wereincubated at 37° C. with 5% CO₂. Cells used for experiments were atpassages 5 (“Low passage”) and 14 (“High passage”).

Scaffolds. Foams composed of 35/65Poly(epsilon-caprolactone)(PCL)/Poly(glycolic acid) (PGA) (35/65 PCUPGA)copolymer, reinforced with Polydioxanone (PDS) mesh (PGA/PCL foam-PDSmesh) were were fromed by the process of lyophilization, as described inU.S. Pat. No. 6,355,699. The foams were 4 cm×5 cm, and 1 mm thick. Foamswere seterilized by treatment with ethylene oxide (ETO). Punches (3.5millimeters) made from scaffolds were loaded with either rhGDF-5 (3.4micrograms/scaffold), TGFbeta-3 (10 nanograms/scaffold), a combinationof rhGDF-5 and TGFbeta-3, or control medium, and lyophilized.

Cell seeding on scaffolds. Placenta- and umbilicus-derived cells weretreated with trypsin, and cell number and viability was determined.7.5×10⁵ cells were resuspended in 15 microliters of Growth Medium andseeded onto 3.5 millimeter scaffold punches in a cell culture dish. Thecell-seeded scaffold was incubated in a cell culture incubator (37° C.,5% CO₂) for 2 hours after which they were placed within cartilageexplant rings.

Bovine Cartilage Explants. Cartilage explants 5 millimeters in diameterwere made from cartilage obtained from young bovine shoulder. Punches (3millimeters) were excised from the center of the explant and replacedwith cells seeded on 3.5 millimeters resorbable scaffold. Scaffolds withcells were retained within the explants using fibrin glue (60microlitersof bovine fibrinogen, 3 milligrams/milliliter). Samples weremaintained in chondrocyte growth medium overnight, rinsed in PhosphateBuffered Saline the following day, and implanted into SCID mice.

Animals. SCID mice ((Mus musculus)/Fox Chase SCID/Male), 5 weeks of age,were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.) andCharles River Laboratories (Portage, Mich.). Animals used in the studywere selected without any apparent systematic bias. A tag was placed oneach individual animal cage listing the accession number, implantationtechnique, animal number, species/strain, surgery date, in vivo period,and date of euthanasia. The animals were identified by sequentialnumbers marked on the ear with an indelible ink marker.

Experimental Design. A total of 42 mice were tested. Two scaffolds wereimplanted subcutaneously in each mouse as described below; 42 mice forsubcutaneous implantation; 28 treatments with n-value of 3 pertreatment. The study corresponds to IACUC Approval Number: SkillmanIACUC 01-037. The study lasted six weeks.

SCID Implantation.

A. Body Weights

Each animal was weighed prior to being anesthetized and at necropsy.

B. Anesthesia and Surgical Preparation:

All handling of the SCID mice occurred under a hood. The mice wereindividually weighed and anesthetized with an intraperitoneal injectionof a mixture of KETASET® (ketamine hydrochloride [60milligram/kilogram]), ROMPUN® (xylazine [10 milligram/kilogram]), andsaline.

After induction of anesthesia, the entire back of the animal from thedorsal cervical area to the dorsal lumbosacral area was clipped free ofhair using electric animal clippers. The area was scrubbed withchlorhexidine diacetate, rinsed with alcohol, dried, and painted with anaqueous iodophor solution of 1% available iodine. Ophthalmic ointmentwas applied to the eyes to prevent drying of the tissue during theanesthetic period. The anesthetized and surgically prepared animal wasplaced in the desired recumbent position.

C. Subcutaneous Implantation Technique:

An approximate 2-centimeters skin incision was made just lateral to thethoracic spine parallel to the vertebral column. The skin was separatedfrom the underlying connective tissue via blunt dissection. Each SCIDmouse received 2 treatments that were placed in subcutaneous pocketscreated by blunt dissection in each hemithorax through one skin incision(Table 26-1) Tacking sutures of 5-0 ETHIBOND EXCEL (polyester) (EthiconInc, Somerville, N.J.) were used to tack the skin to musculature aroundeach scaffold to prevent subcutaneous migration. Scaffolds wereimplanted for 6 weeks and then harvested. The experimental design isoutlined in Table 26-1. TABLE 26-1 Experimental Design: Treatment (N = 3per treatment) A. 65/35 PGA/PCL Foam + PDS mesh cultured with Placentalderived cells, EP, TGFb3 B. 65/35 PGA/PCL Foam + PDS mesh cultured withPlacental derived cells, EP, rhGDF-5 C. 65/35 PGA/PCL Foam + PDS meshcultured with Placental derived cells, EP, rhGDF- 5 + TGFb3 D. 65/35PGA/PCL Foam + PDS mesh cultured with Placental derived cells, EP,control E. 65/35 PGA/PCL Foam + PDS mesh cultured with Placental derivedcells, LP, TGFb3 F. 65/35 PGA/PCL Foam + PDS mesh cultured withPlacental derived cells, LP, rhGDF-5 G. 65/35 PGA/PCL Foam + PDS meshcultured with Placental derived cells, LP, rhGDF- 5 + TGFb3 H. 65/35PGA/PCL Foam + PDS mesh cultured with Placental derived cells, LP,control I. 65/35 PGA/PCL Foam + PDS mesh cultured with Umbilical derivedcells, EP, TGFb3 J. 65/35 PGA/PCL Foam + PDS mesh cultured withUmbilical derived cells, EP, rhGDF-5 K. 65/35 PGA/PCL Foam + PDS meshcultured with Umbilical derived cells, EP, rhGDF- 5 + TGFb3 L. 65/35PGA/PCL Foam + PDS mesh cultured with Umbilical derived cells, EP,control M. 65/35 PGA/PCL Foam + PDS mesh cultured with Umbilical derivedcells, LP, TGFb3 N. 65/35 PGA/PCL Foam + PDS mesh cultured withUmbilical derived cells, LP, rhGDF-5 O. 65/35 PGA/PCL Foam + PDS meshcultured with Umbilical derived cells, LP, rhGDF- 5 + TGFb3 P. 65/35PGA/PCL Foam + PDS mesh cultured with Umbilical derived cells, LP,control Q. 65/35 PGA/PCL Foam + PDS mesh cultured with hMSC, TGFb3 R.65/35 PGA/PCL Foam + PDS mesh cultured with hMSC, rhGDF-5 S. 65/35PGA/PCL Foam + PDS mesh cultured with hMSC, rhGDF-5 + TGFb3 T. 65/35PGA/PCL Foam + PDS mesh cultured with hMSC, control U. 65/35 PGA/PCLFoam + PDS mesh cultured with fibroblasts, Adult TGFb3 V. 65/35 PGA/PCLFoam + PDS mesh cultured with fibroblasts, Adult rhGDF-5 W. 65/35PGA/PCL Foam + PDS mesh cultured with fibroblasts, Adult rhGDF-5 + TGFb3X. 65/35 PGA/PCL Foam + PDS mesh cultured with fibroblasts, Adultcontrol Y. 65/35 PGA/PCL Foam + PDS mesh, TGFb3 Z. 65/35 PGA/PCL Foam +PDS mesh, rhGDF-5 AA. 65/35 PGA/PCL Foam + PDS mesh, rhGDF-5 + TGFb3 BB.65/35 PGA/PCL Foam + PDS mesh, control

D. Necropsy and Histologic Preparation

Gross examination was performed on any animals that died during thecourse of the study or were euthanized in moribund condition. Selectedtissues were saved at the discretion of the study director and/orpathologist.

Mice were euthanized by CO₂ inhalation at their designated intervals.Gross observations of the implanted sites were recorded. Samples of thesubcutaneous implantation sites with their overlying skin were excisedand fixed in 10% buffered formalin. Each implant was bisected intohalves, and one half was sent to MPI Research (Mattawan, Mich.) forparaffin embedding, sectioning, and staining with Hematoxylin & Eosin(H&E) and Safranin O (SO).

Results

New cartilage and bone formation was observed in the majority of thesamples including growth factor-loaded, cell-seeded scaffolds,cell-seeded control scaffolds, and scaffolds loaded with growth factoralone. The extent of new cartilage and bone formation varied within thetreatment and control groups.

Early and Late passage placenta-derived cell seeded scaffolds showed newcartilage and bone formation within the scaffolds. No obviousdifferences in new cartilage and bone formation was observed between thedifferent growth factor-loaded, cell-seeded scaffolds and scaffoldsseeded with cells alone. Compared to control scaffolds (without growthfactors and without cells), it appeared that there was greater extent ofnew cartilage formation in cell-seeded scaffolds both with and withoutgrowth factors and in growth factor-loaded scaffolds alone. Newcartilage formation with placenta-derived cell-seeded scaffolds wassimilar to MSC- and fibroblast-seeded scaffolds.

In growth factor-treated and control scaffolds seeded withumbilicus-derived cells at early and late passage, new cartilage andbone formation were observed. The extent of cartilage formation appearedto be less than that seen with placenta-derived cells. No one sampleshowed extensive cartilage formation as seen with the placenta-derivedcells. Bone formation appeared to be higher in scaffolds seeded withumbilicus-derived cells on scaffolds containing both TGFbeta-3 andrhGDF-5.

hMSC-loaded scaffolds also showed new cartilage and bone formation. Theextent of new cartilage and bone formation was similar for all the hMSCtreatment groups. Human adult fibroblast seeded scaffolds alsodemonstrated new cartilage and bone formation. Results were similar tothose obtained with placenta-derived cells and hMSCs

In the control group, in which growth factor-loaded scaffolds orscaffold alone were placed in cartilage rings and implanted, newcartilage and bone formation were also observed. Not surprisingly, theextent of new cartilage formation was greater in scaffolds with growthfactor than in scaffolds without growth factor. Increased bone formationwas present in the control with the combination of the two tested growthfactors.

New cartilage formation was observed adjacent to the cartilage explantrings as well as within the scaffolds. New cartilage formation withinthe scaffolds adjacent to the cartilage rings could be a result ofchondrocyte migration. Cartilage formation seen as islands within thescaffolds may be a result of either migration of chondrocytes within thescaffolds, differentiation of seeded cells or differentiation ofendogenous mouse progenitor cells. This observation stems from the factthat in control growth factor-loaded scaffolds with no seeded cells,islands of chondrogenic differentiation were observed. New boneformation was observed within the scaffolds independently and alsoassociated with chondrocytes. Bone formation may have arisen fromosteoblast differentiation as well as endochondral ossification.

It is difficult to separate new cartilage and bone formation associatedwith chondrocytes that migrated versus that from any chondrogenic andosteogenic differentiation of seeded cells that may have occurred.Staining of sections with specific human antibodies may distinguish thecontribution of the seeded cells to the observed chondrogenesis andosteogenesis. It is also possible that placenta-derived cells andumbilicus-derived cells stimulated chondrocyte migration.

Abundant new blood vessels were observed with the scaffolds loaded withplacenta-derived cells and umbilicus-derived cells. Blood vessels wereabundant in areas of bone formation. New blood vessels were alsoobserved within the hMSC- and fibroblast-seeded scaffolds associatedwith new bone formation.

Systemic effects of the adjacent scaffold (with growth factor (GF)) onthe control scaffolds (no GF, no cells) on promoting new cartilage andbone formation cannot be ruled out. Analysis of new cartilage and boneformation in scaffolds, taking into consideration the scaffoldsimplanted adjacent to it in SCID mice, showed no clear pattern ofsystemic effect of growth factor from the adjacent scaffold.

Summary. Results showed that new cartilage and bone formation wereobserved in growth factor and control scaffolds seeded with placenta-and umbilicus-derived cells. Results with placenta-derived cells weresimilar to that seen with human mesenchymal stem cells, while the extentof new cartilage like tissue formation was slightly less pronounced inumbilicus-derived cells. Growth factor-loaded scaffolds implantedwithout cells also demonstrated new cartilage and bone formation. Thesedata indicate that new cartilage formation within the scaffolds mayarise from chondrocytes that migrated from the bovine explants, fromchondrogenic differentiation of endogenous progenitor cells, and fromchondrogenic differentiation of seeded cells.

These results suggest that placenta- and umbilicus-derived cells undergochondrogenic and osteogenic differentiation. These results also suggestthat placenta- and umbilicus-derived cells may promote migration ofchondrocytes from the cartilage explant into the scaffolds. Abundant newblood vessels were also observed in the scaffolds especially associatedwith new bone formation.

Biological Deposit of Umbilicus-Derived Cells and Cultures

Consistent with the detailed description and the written examplesprovided herein, examples of umbilicus-derived cells of the inventionwere deposited with the American Type Culture Collection (ATCC,Manassas, Va.) on Jun. 10, 2004, and assigned ATCC Accession Numbers asfollows: (1) strain designation UMB 022803 (P7) was assigned AccessionNo. PTA-6067; and (2) strain designation UMB 022803 (P17) was assignedAccession No. PTA-6068.

While the present invention has been particularly shown and describedwith reference to the presently preferred embodiments, it is understoodthat the invention is not limited to the embodiments specificallydisclosed and exemplified herein. Numerous changes and modifications maybe made to the preferred embodiment of the invention, and such changesand modifications may be made without departing from the scope andspirit of the invention as set forth in the appended claims.

1. An isolated human umbilicus-derived cell capable of self-renewal andexpansion in culture and having the potential to differentiate intocells of other phenotypes, wherein the cell does not substantiallystimulate naïve CD4⁺ T cells, and expresses PD-L2, but not HLA-G, CD178, HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, or B7-H2.
 2. The isolated cellof claim 1 wherein the cell produces vimentin and alpha-smooth muscleactin.
 3. The isolated cell of claim 1 wherein the cell secretes one ormore cellular factors.
 4. The isolated cell of claim 3 wherein thefactors are MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO,or TIMP1.
 5. The isolated cell of claim 4 wherein the cell secretes eachof MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, andTIMP1.
 6. The isolated cell of claim 1 wherein the cell does not secreteone or more of the cellular factors SDF-1alpha, TGF-beta2, ANG2, PDGFbbor VEGF, as detected by ELISA.
 7. The isolated cell of claim 6, whereinthe cell secretes one or more of the factors MCP-1, IL-6, IL-8, GCP-2,HGF, KGF, FGF, HB-EGF, BDNF, TPO, and TIMP1.
 8. The isolated cell ofclaim 7 wherein the cell secretes each of the factors MCP-1, IL-6, IL-8,GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, and TIMP1.
 9. The isolated cellof claim 6 wherein the cell does not secrete any of the factorsSDF-1alpha, TGF-beta2, ANG2, PDGFbb or VEGF, as detected by ELISA. 10.The isolated cell of claim 9, wherein the cell secretes one or more ofthe factors MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO,or TIMP1.
 11. The isolated cell of claim 10 wherein the cell secreteseach of the factors MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF,BDNF, TPO, and TIMP1.
 12. The isolated cell of claim 7 wherein the celldoes not secrete any of the factors SDF-1alpha, TGF-beta2, ANG2, PDGFbbor VEGF, as detected by ELISA.
 13. An injectable therapeutic cellcomprising an isolated human umbilicus-derived cell of claim
 1. 14. Theinjectable cell of claim 13 treated to inactivate tissue factor.
 15. Theinjectable cell of claim 14 wherein the treated is with an anti-tissuefactor antibody.